Methods and compositions for treating malignant tumors associated with kras mutation

ABSTRACT

This invention provides methods and compositions for preventing, treating or ameliorating one or more symptoms of a malignant tumor, which may be associated with KRAS mutation in a mammal in need thereof, by administering to the mammal a therapeutically effective amount of a composition comprising one or more RNAi molecules that are active in reducing expression of GST-π.

SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asan ASCII file created on Jan. 2, 2016, named ND7023946US_SL.txt, whichis 120,619 bytes in size, and is hereby incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

Glutathione S-transferases (IUBMB EC 2.5.1.18) are a family of enzymesthat play an important role in detoxification by catalyzing theconjugation of many hydrophobic and electrophilic compounds with reducedglutathione. Based on their biochemical, immunologic, and structuralproperties, the soluble GSTs are categorized into four main classes:alpha, mu, pi, and theta. Some of these forms are suggested to act toprevent carcinogenesis by detoxifying proximate or ultimate carcinogens,especially electrophilic agents including Michael reaction acceptors,diphenols, quinones, isothiocyanates, peroxides, vicinal dimercaptans,etc. However, in neoplastic cells, specific forms are known to beexpressed and have been known to participate in their resistance toanticancer drugs.

The glutathione S-transferase-π gene (GSTP1) is a polymorphic geneencoding active, functionally different GSTP1 variant proteins that arethought to function in xenobiotic metabolism and play a role insusceptibility to cancer. It is expressed abundantly in tumor cells.See, e.g., Aliya S. et al. Mol Cell Biochem., 2003 November;253(1-2):319-327. Glutathione S-transferase-P is an enzyme that inhumans is encoded by the GSTP1 gene. See, e.g., Bora P S, et al.(October 1991) J. Biol. Chem., 266 (25): 16774-16777. The GST-πisoenzyme has been shown to catalyze the conjugation of GSH with somealkylating anti-cancer agents, suggesting that over-expression of GST-πwould result in tumor cell resistance.

Elevated serum GST-π levels were observed in patients with variousgastrointestinal malignancies including gastric, esophageal, colonic,pancreatic, hepatocellular, and biliary tract cancers. Patients withbenign gastrointestinal diseases had normal GST-π, but some patientswith chronic hepatitis and cirrhosis had slightly elevated levels. Over80% of patients with Stage III or IV gastric cancer and even about 50%of those with Stage I and II had elevated serum GST-π. See, e.g., NiitsuY, et al. Cancer, 1989 Jan. 15; 63(2):317-23. Elevated GST-π levels inplasma were observed in patients with oral cancer, but patients withbenign oral diseases had normal GST-π levels. GST-π was found to be auseful marker for evaluating the response to chemotherapy, formonitoring postoperative tumor resectability or tumor burden, and forpredicting the recurrence of tumor in patients with oral cancer. See,e.g., Hirata S. et al. Cancer, 1992 November 15:70(10):2381-7.

Immunohistochemical studies have revealed that many cancers,histologically classified as adenocarcinomas or squamous cellcarcinomas, express GST-71 Plasma or serum GST-π levels are increased in30-50% of patients with cancers of the gastrointestinal tract. This formis also suggested to participate in resistance to anticancer drugs suchas cisplatin and daunorubicin, and its expression in cancer tissues maybe of prognostic value in cancer patients.

The protein product of the normal human KRAS gene (V-Ki-ras2 Kirsten ratsarcoma viral oncogene homolog) performs a signaling function in normaltissue, and the mutation of a KRAS gene is a putative step in thedevelopment of many cancers. See, e.g. Kranenburg O, November 2005,Biochim. Biophys. Acta, 1756(2):81-82. The KRAS protein is a GTPase andis involved in several signal transduction pathways. KRAS acts as amolecular on/off switch which activates proteins necessary for thepropagation of growth factor and signals of other receptors such asc-Raf and PI 3-kinase.

Mutation in KRAS can be related to malignant tumors, such as lungadenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas, andcolorectal carcinoma. In human colorectal cancer, KRAS mutation appearsto induce overexpression of GST-π via activation of AP-1. See, e.g.,Miyanishi et al., Gastroenterology, 2001; 121 (4):865-74.

Mutant KRAS is found in colon cancer (Burmer G C, Loeb L A, 1989, Proc.Natl. Acad. Sci. U.S.A., 86(7):2403-2407), pancreatic cancer (AlmogueraC, et al., 1988, Cell, 53(4):549-554) and lung cancer (Tam I Y, et al.,2006, Clin. Cancer Res., 12(5):1647-1653). KRAS accounts for 90% of RASmutations in lung adenocarcinomas (Forbes S, et al. Cosmic 2005. Br JCancer, 2006; 94:318-322).

KRAS gene may also be amplified in colorectal cancer. KRAS amplificationcan be mutually exclusive with KRAS mutations. See, e.g., Valtorta E, etal., 2013, Int. J. Cancer, 133(5):1259-65. Amplification of wild-typeKRAS also has been observed in ovarian, gastric, uterine, and lungcancers. See, e.g., Chen Y, et al., 2014, PLoS ONE, 9(5):e98293.

Expression of GST-π increases in various cancer cells, which may berelated to resistance to some anticancer agents. See, e.g. Ban et al.,Cancer Res., 1996, 56(15):3577-82; Nakajima et al., J Pharmacol ExpTher., 2003, 306(3):861-9.

Agents for suppressing GST-π have been disclosed for inducing apoptosisin cells. However, such compositions and techniques also causedautophagy and required the combined action of various agents. See, e.g.,US 2014/0315975 A1. Moreover, suppressing GST-π has not been found toshrink or reduce tumors. For example, in a cancer that wasoverexpressing GST-π, the weights of tumors were not affected bysuppressing GST-π, although other effects were observed. See, e.g.,Hokaiwado et al., Carcinogenesis, 2008, 29(6):1134-1138.

There is an urgent need for methods and compositions to developtherapies for patients with KRAS associated malignancies.

What is needed are methods and compositions for preventing or treatingmalignant tumors. There is a continuing need for RNAi molecules, andother structures and compositions for preventing, treating, reducing orshrinking malignant tumors.

BRIEF SUMMARY

This invention relates to the fields of biopharmaceuticals andtherapeutics composed of nucleic acid based molecules. Moreparticularly, this invention relates to tumor therapies for preventing,treating or ameliorating KRAS-associated cancers in which the cancercells contain a KRAS mutation or display aberrant KRAS expressionlevels. This invention further relates to a pharmaceutical compositioncontaining one or more RNAi molecules for inhibiting expression ofGST-π.

This invention relates to the surprising discovery that malignant tumorsize can be reduced in vivo by treatment with siRNA inhibitors of GST-π.

In some embodiments, malignant tumors containing a KRAS mutation ordisplaying aberrant KRAS expression levels can be reduced by treatmentwith siRNA agents that modulate expression of GST-π.

This invention relates to methods and compositions for nucleic acidbased therapeutic compounds against malignant tumors. In someembodiments, this invention provides RNAi molecules, structures andcompositions that can silence expression of GST-π. The structures andcompositions of this disclosure can be used in preventing, treating orreducing the size of malignant tumors.

This invention provides compositions and methods that may be used fortreating a neoplasia in a subject. In particular, this inventionprovides therapeutic compositions that can decrease the expression of aGST-π nucleic acid molecule or polypeptide for treating aKRAS-associated neoplasia without unwanted autophagy.

In some aspects, this invention includes an inhibitory nucleic acidmolecule that corresponds to, or is complementary to at least a fragmentof a GST-π nucleic acid molecule, and that decreases GST-π expression ina cell.

In further aspects, the invention features a double-stranded inhibitorynucleic acid molecule that corresponds to, or is complementary to atleast a fragment of a GST-π nucleic acid molecule that decreases GST-πexpression in a cell. In certain embodiments, the double-strandednucleic acid molecule is a siRNA or a shRNA.

In some aspects, this invention includes a vector encoding an inhibitorynucleic acid molecule described above. A vector can be a retroviral,adenoviral, adeno-associated viral, or lentiviral vector. In furtherembodiments, a vector can contain a promoter suitable for expression ina mammalian cell. Additional embodiments include cancer cells containinga KRAS mutation or displaying aberrant KRAS expression levels, which canalso contain the vector, or an inhibitory nucleic acid molecule of anyone of the above aspects. In further embodiments, the cells can beneoplastic cells in vivo.

In some embodiments, this invention includes methods for decreasingGST-π expression in a malignant tumor cell containing a KRAS mutation ordisplaying aberrant KRAS expression. Methods can include contacting thecell with an effective amount of an inhibitory nucleic acid moleculecorresponding to, or complementary to at least a portion of a GST-πnucleic acid molecule, where the inhibitory nucleic acid moleculeinhibits expression of a GST-π polypeptide, thereby decreasing GST-πexpression in the cell.

In certain embodiments, the inhibitory nucleic acid molecule can be anantisense nucleic acid molecule, a small interfering RNA (siRNA), or adouble-stranded RNA (dsRNA) that is active for inhibiting geneexpression.

In additional embodiments, methods of this invention can decrease GST-πtranscription or translation in malignant tumors.

In particular embodiments, this invention includes methods fordecreasing GST-π expression in a malignant tumor cell, where the cellcan be a human cell, a neoplastic cell, a cell in vivo, or a cell invitro.

Embodiments of this invention can also provide methods for treating asubject having a neoplasm, where neoplasm cancer cells contain a KRASmutation or display aberrant KRAS expression levels. Methods can involveadministering to the subject an effective amount of an inhibitorynucleic acid molecule corresponding to, or complementary to a GST-πnucleic acid molecule, where the inhibitory nucleic acid moleculereduces GST-π expression, thereby treating the neoplasm. In someembodiments, methods of this invention can decrease the size of aneoplasm, relative to the size of the neoplasm prior to treatment orwithout treatment.

In various embodiments, an inhibitory nucleic acid molecule can bedelivered in a liposome, a polymer, a microsphere, a nanoparticle, agene therapy vector, or a naked DNA vector.

In further aspects, this invention features methods for treating asubject, e.g. a human patient, having a neoplasm in which the neoplasmcancer cells contain a KRAS mutation or display aberrant KRAS expressionlevels. In certain embodiments, the methods can include administering tothe subject an effective amount of an inhibitory nucleic acid molecule,where the inhibitory nucleic acid molecule is an antisense nucleic acidmolecule, a siRNA, or a dsRNA that inhibits expression of a GST-πpolypeptide.

In particular embodiments, a cell of the neoplasm overexpresses GST-π.

In certain embodiments, the neoplasm can be a malignant tumor, or lungcancer, or pancreatic cancer.

Embodiments of this invention include the following:

A pharmaceutical composition for the treatment or therapy of a tumorassociated with a mutation in the KRAS gene or overexpression ofwild-type KRAS gene, the composition comprising RNAi molecules andpharmaceutically acceptable excipients, wherein the RNAi moleculescomprise a nucleotide sequence corresponding to a target sequence ofGST-π.

In some embodiments, the pharmaceutical composition includes RNAimolecules that have a duplex region comprising a nucleotide sequencecorresponding to a target sequence of GST-π mRNA.

In certain aspects, the RNAi molecules are siRNAs or shRNAs that areactive for suppressing gene expression.

The pharmaceutical composition can include pharmaceutically acceptableexcipients such as one or more lipid compounds. The lipid compounds mayinclude lipid nanoparticles. In certain embodiments, the lipidnanoparticles can encapsulate the RNAi molecules.

This invention further contemplates methods for preventing, treating orameliorating one or more symptoms of a malignant tumor associated withKRAS mutation in a mammal in need thereof, the method comprising:

identifying a tumor cell in the mammal, the tumor cell comprising atleast one of: (i) a mutation of the KRAS gene, and (ii) an aberrantexpression level of KRAS protein; and

administering to the mammal a therapeutically effective amount of acomposition comprising one or more RNAi molecules that are active inreducing expression of GST-π.

In such methods, the mammal can be a human, and the GST-π can be a humanGST-π. The RNAi molecule can be a siRNA, shRNA, or microRNA.

In certain embodiments, the RNAi molecule can have a duplex region,wherein the duplex region can include a nucleotide sequencecorresponding to a target sequence of GST-π mRNA. The RNAi molecule candecrease expression of GST-π in the mammal.

In some embodiments, the administration can decrease expression of GST-πin the mammal by at least 5% for at least 5 days. In certainembodiments, the administration can decrease the volume of the malignanttumor in the mammal by at least 5%, or at least 10%, or at least 20%, orat least 30%, or at least 40%, or at least 50%. In additionalembodiments, the method can reduce one or more symptoms of the malignanttumor, or delay or terminate progression or growth of the malignanttumor.

In certain embodiments, the administration can reduce growth ofmalignant tumor cells in the subject. The administration can reducegrowth for at least 2%, or at least 5%, or at least 10%, or at least15%, or at least 20% of the malignant tumor cells in the subject.

In general, the tumor cells can have increased levels of expression ofwild type KRAS protein compared to that in a normal cell. In someembodiments, the tumor cell over-express wild-type GST-π RNA or protein.

In particular, the tumor cell can have mutations in the KRAS protein atone or more of residues 12, 13 and 61.

This invention contemplates that the tumor cell can have mutations inthe KRAS protein, and the tumor can be a cancer selected from lungcancer, colon cancer, and pancreatic cancer.

In some embodiments, the tumor cell can have mutations in the KRASprotein, and the tumor can be a sarcoma selected from the groupconsisting of lung adenocarcinoma, mucinous adenoma, ductal carcinoma ofthe pancreas, and colorectal carcinoma. In certain embodiments, themalignant tumor can be a sarcoma selected from the group of lungadenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas,colorectal carcinoma, breast cancer, and fibrosarcoma. Also, themalignant tumor can be located in an anatomical region selected from thegroup of lung, colon, pancreas, gallbladder, liver, breast, and anycombination thereof.

Aspects of this invention can provide methods in which theadministration is performed from 1 to 12 times per day. Theadministration can be performed for a duration of 1, 2, 3, 4, 5, 6 or 7days. In certain embodiments, the administration can be performed for aduration of 1, 2, 3, 4, 5, 6, 8, 10 or 12 weeks.

A dose for administration can be from 0.01 to 2 mg/kg of the RNAimolecules at least once per day for a period up to twelve weeks. In someembodiments, the administration can provide a mean AUC(0-last) of from 1to 1000 ug*min/mL and a mean C_(max) of from 0.1 to 50 ug/mL for theGST-π RNAi molecule.

The administration can be by intravenous injection, intradermalinjection, subcutaneous injection, intramuscular injection,intraperitoneal injection, oral, topical, infusion, or inhaled.

These and other aspects will become apparent from the followingdescription of the embodiments taken in conjunction with the followingdrawings, although variations and modifications therein may be affectedwithout departing from the spirit and scope of the novel concepts of thedisclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the profound reduction of orthotopic lung cancer tumors invivo by a siRNA of this invention targeted to GST-π. The GST-π siRNA wasadministered in a liposomal formulation at a dose of 2 mg/kg to athymicnude mice presenting A549 orthotopic lung cancer tumors. Final primarytumor weights were measured at necropsy for the treatment group and avehicle control group. The GST-π siRNA showed significant efficacy forinhibition of lung cancer tumors in this six-week study. As shown inFIG. 1, after 43 days, the GST-π siRNA showed markedly advantageoustumor inhibition, with final primary tumor average weights significantlyreduced by 2.8-fold, as compared to control.

FIG. 2 shows tumor inhibition efficacy in vivo for a GST-π siRNA. Acancer xenograft model using A549 cells was utilized with a relativelylow dose of siRNA at 0.75 mg/kg. The GST-π siRNA showed advantageoustumor inhibition within a few days. After 36 days, the GST-π siRNAshowed markedly advantageous tumor inhibition, with final tumor averagevolumes significantly reduced by about 2-fold, as compared to control.

FIG. 3 shows tumor inhibition efficacy in vivo for a GST-π siRNA at theendpoint of FIG. 2. The GST-π siRNA showed advantageous tumor inhibitionwith average tumor weights reduced by more than 2-fold.

FIG. 4 shows that a GST-π siRNA of this invention greatly increasedcancer cell death by apoptosis in vitro. The GST-π siRNA causedupregulation of PUMA, a biomarker for apoptosis, which is associatedwith loss in cell viability. In FIG. 4, the expression of PUMA wasgreatly increased from 2-6 days after transfection of the GST-π siRNA.

FIG. 5 shows that a GST-π siRNA of this invention provided knockdownefficacy for A549 xenograft tumors in vivo. Dose dependent knockdown ofGST-π mRNA was observed in athymic nude (nu/nu) female mice (CharlesRiver) with the siRNA targeted to GST-π. As shown in FIG. 5, at a doseof 4 mg/kg, significant reduction of about 40% in GST-π mRNA wasdetected 24 hours after injection.

FIG. 6 shows that a GST-π siRNA of this invention inhibited pancreaticcancer xenograft tumors in vivo. The GST-π siRNA provided gene silencingpotency in vivo when administered in a liposomal formulation topancreatic cancer xenograft tumors in athymic nude female mice, 6 to 8weeks old. As shown in FIG. 6, a dose response was obtained with dosesranging from 0.375 mg/kg to 3 mg/kg of siRNA targeted to GST-π. TheGST-π siRNA showed advantageous tumor inhibition within a few days afteradministration, the tumor volume being reduced by about 2-fold at theendpoint.

FIG. 7 shows that a GST-π siRNA of this invention exhibited increasedserum stability. As shown in FIG. 7, the half-life (t_(1/2)) in serumfor both the sense strand (FIG. 7, top) and antisense strand (FIG. 7,bottom) of a GST-π siRNA was about 100 minutes.

FIG. 8 shows that a GST-π siRNA of this invention exhibited enhancedstability in formulation in plasma. FIG. 8 shows incubation of aliposomal formulation of a GST-π siRNA in 50% human serum in PBS, anddetection of remaining siRNA at various time points. As shown in FIG. 8,the half-life (t_(1/2)) in plasma of the formulation of the GST-π siRNAwas significantly longer than 100 hours.

FIG. 9 shows in vitro knockdown for the guide strand of a GST-π siRNA.As shown in FIG. 9, the guide strand knockdown of the GST-π siRNA wasapproximately exponential, as compared to a control with scrambledsequence that exhibited no effect.

FIG. 10 shows in vitro knockdown for the passenger strand of the GST-πsiRNA of FIG. 9. As shown in FIG. 10, the passenger strand off targetknockdown for the GST-π siRNA was greatly reduced, with essentially noeffect.

FIG. 11 shows in vitro knockdown for the guide strands of several highlyactive GST-π siRNAs. As shown in FIG. 11, the guide strand knockdownactivities of the GST-π siRNAs were approximately exponential.

FIG. 12 shows in vitro knockdown for the passenger strand of the GST-πsiRNAs of FIG. 11. As shown in FIG. 12, the passenger strand off targetknockdown activities for the GST-π siRNAs were significantly reducedbelow about 500 pM.

FIG. 13 shows in vitro knockdown for the guide strand of a highly activeGST-π siRNA. As shown in FIG. 13, the guide strand knockdown activity ofthe GST-π siRNA was approximately exponential.

FIG. 14 shows in vitro knockdown for the passenger strand of the GST-πsiRNA of FIG. 13. As shown in FIG. 14, the passenger strand off targetknockdown activity for the GST-π siRNA was significantly reduced.

FIG. 15 shows tumor inhibition efficacy in vivo for GST-π siRNAs havingstructure based on siRNA A9. A cancer xenograft model using A549 cellswas utilized with a relatively low dose of siRNA at 0.5 mg/kg. The GST-πsiRNAs showed advantageous tumor inhibition within a few days. After 36days, the GST-π siRNAs showed markedly advantageous tumor inhibition,with final tumor average volumes significantly reduced by about 2-fold,as compared to control.

FIG. 16 shows tumor inhibition efficacy in vivo for a GST-π siRNA havingstructure based on siRNA B13. A cancer xenograft model using A549 cellswas utilized with a relatively low dose of siRNA at 0.75 mg/kg. TheGST-π siRNA showed advantageous tumor inhibition within a few days.After 36 days, the GST-π siRNA showed markedly advantageous tumorinhibition, with final tumor average volumes significantly reduced byabout 2-fold, as compared to control.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods for utilizing therapeutic compositionsthat decrease the expression of a GST-π nucleic acid molecule orpolypeptide for the treatment of a neoplasia in a subject, wherein theneoplasia is associated with cells containing a KRAS mutation ordisplaying aberrant KRAS expression levels.

The therapeutic compositions of this invention can include inhibitorynucleic acid molecules such as siRNAs, shRNAs, and antisense RNAs.

GST-π denotes an enzyme, which is encoded by the GSTP1 gene, andcatalyzes glutathione conjugation. GST-π is present in various animals,including humans, and its sequence information is known and given inNCBI database accession numbers (e.g., human: NP_000843 (NM_000852),rat: NP_036709 (NM_012577), mouse: NP_038569 (NM_013541), etc.

By “GST-π polypeptide” is meant a protein or protein variant, orfragment thereof, that is substantially identical to at least a portionof a protein encoded by the GST-π coding sequence. By “GST-π nucleicacid molecule” is meant a polynucleotide encoding a GST-π polypeptide orvariant, or fragment thereof.

Occurrence of a mutation of a gene sequence or an amino acid sequencebetween biological individuals may not impair the physiological functionof a protein. GST-π and GSTP1 gene in this invention are not limited toa protein or nucleic acid having the same sequence as the GST-πsequences listed herein, and can include those that have a sequence thatis different from the above sequence by one or more amino acids orbases, for example, one, two, three, four, five, six, seven, eight,nine, or ten amino acids or bases, but have an equivalent function tothat of the known GST-π.

The sequence of Human glutathione S-transferase gene (GST-π), completeCDS, GenBank Accession No.: U12472, is shown in Table 1.

TABLE 1 The complete sequence of the human GSTπ gene. (SEQ ID NO: 1) 1gtggctcacc tgtacccagc acttgggaag ccgaggcgtg cagatcacct aagtcaggag 61ttcgagacca gcccggccaa catggtgaaa ccccgtctct actaaaaata caaaaatcag 121ccagatgtgg cacgcaccta tatccaccta ctcgggaggc tgaagcagaa tgcttaaccc 181gagaggcgga ggttgcagtg agccgcccag atcgcgccac tgcactccag cctgggccac 241agcgtgagac tactcataaa ataaaataaa ataaaataaa ataaaataaa ataaaataaa 301ataataaaat aaaataaaat aaaataaaat ataaaataaa ataaaataaa ataaaataaa 361ataaaataaa ataaaagcaa tttcctttcc tctaagcggc ctccacccct ctcccctgcc 421ctgtgaacgg gggaagctcc ggatcgcagc aattagggaa tttccccccg cgatgtcccg 481gcgcgccagt tcggcgcaca tctttcgctg cggtcctctt cctgctgtct gtttactccc 541taggcccctg gacctgggaa agagggaaag gcttcccgcc agctgcgcgg cgactccggg 601gactccaggg cgcccctctg cggcgacgcc cgggtgcagc ggccgccggg ctggggccgg 661cgggactccg cgggaccctc cagaagagcg gccggcggct gactcagcac tggggcggag 721gggcgggaca cccttataag gctcggagcg cgagccttcg ctggagtttc gccgccgcag 781tcttcgccac cagtgagtac gcggccgcgt ccccggggat ggggctcaga gctccagcat 841ggggccaacc cgcagcatca ggccgggctc ccggcggcct ccccacctcg agacccggga 901cggggcctag gggacccagg acgtcccagt gccgttagcg gctttcaggg ggcccggagc 961gcctcgggga gggatgggac cccgggggcg ggagggcagc tcactcaccg cgccttggca 1021tcctccccgg gctccacaaa ttttctttgt tcgctgcagt gccgccctac accgtggtct 1081atttcccagt tcgaggtagg agcatgtgtc tggcagggaa gggaggcagg ggctggggct 1141gcagcaccca cagcccccac ccggagagat ccgaaccccc ttatccctcg tcgtgtgctt 1201ttacccccgg cctccttcct gttccccgcc tctcccgcca tgcctgctcc ccgccccagt 1261gttgtgtgaa atcttcggag gaacctgttt ccctgttccc tccctgcact cctgacccct 1321ccccgggttg ctgcgaggcg gagtcggccc ggtccccaca tctcgtactt ctccctcccc 1381gcaggccgct gcgcggccct gcgcatgctg ctggcagatc agggccagag ctggaaggag 1441gaggtggtga ccgtggagac gtggcaggag ggctcactca aagcctcctg cgtaagtgac 1501catgcccggg caaggggagg gggtgctggg ccttaggggg ctgtgactag gatcggggga 1561cgccccaagc tcagtgcccc tccctgagcc atgcctcccc caacagctat acgggcagct 1621ccccaagttc caggacggag acctcaccct gtaccagtcc aataccatcc tgcgtcacct 1681gggccgcacc cttggtgagt cttgaacctc caagtccagg gcaggcatgg gcaagcctct 1741gcccccggag cccttttgtt taaatcagct gccccgcagc cctctggagt ggaggaaact 1801gagacccact gaggttacgt agtttgccca aggtcaagcc tgggtgcctg caatccttgc 1861cctgtgccag gctgcctccc aggtgtcagg tgagctctga gcacctgctg tgtggcagtc 1921tctcatcctt ccacgcacat cctcttcccc tcctcccagg ctggggctca cagacagccc 1981cctggttggc ccatccccag tgactgtgtt gatcaggcgc ccagtcacgc ggcctgctcc 2041cctccaccca accccagggc tctatgggaa ggaccagcag gaggcagccc tggtggacat 2101ggtgaatgac ggcgtggagg acctccgctg caaatacatc tccctcatct acaccaacta 2161tgtgagcatc tgcaccaggg ttgggcactg ggggctgaac aaagaaaggg gcttcttgtg 2221ccctcacccc ccttacccct caggtggctt gggctgaccc cttcttgggt cagggtgcag 2281gggctgggtc agctctgggc caggggggcc tgggacaaga cacaacctgc acccttattg 2341cctgggacat caaccaccca agtaacgggt catgggggcg agtgcaagga cagagacctc 2401cagcaactgg tggtttctgc tctcctgggg tggccagagg tggaggagga tttgtgccag 2461tttctggatg gagccgctgg cgcttttagc tgaggaaaat atgagacaca gagcactttg 2521ggtaccaggg accagttcag cagaggcagc gtgtgtggcg tgtgtgtgcg tgtgtgtgcg 2581tgtgtgtgtg tacgcttgca tttgtgtcgg gtgggtaagg agatagagat ggggcggcag 2641taggcccagg tcccgaaggc cttgaaccca ctggtttgga gtctcctaag ggcaatgggg 2701gccattgaga agtctgaaca gggctgtgtc tgaatgtgag gtctagaagg atcctccaga 2761gaagccagct ctaaagcttt tgcaatcatc tggtgagaga acccagcaag gatggacagg 2821cagaatggaa tagagatgag ttggcagctg aagtggacag gatttggtac tagcctggtt 2881gtggggagca agcagaggag aatctgggac tctggtgtct ggcctggggc agacgggggt 2941gtctcagggg ctgggaggga tgagagtagg atgatacatg gtgtgtgctg gcaggaggcg 3001ggcaaggatg actatgtgaa ggcactgccc gggcaactga agccttttga gaccctgctg 3061tcccagaacc agggaggcaa gaccttcatt gtgggagacc aggtgagcat ctggccccat 3121gctgttcctt cctcgccacc ctctgcttcc agatggacac aggtgtgagc catttgttta 3181gcaaagcaga gcagacctag gggatgggct taggccctct gcccccaatt cctctccagc 3241ctgctcccgc tggctgagtc cctagccccc ctgccctgca gatctccttc gctgactaca 3301acctgctgga cttgctgctg atccatgagg tcctagcccc tggctgcctg gatgcgttcc 3361ccctgctctc agcatatgtg gggcgcctca gtgcccggcc caagctcaag gccttcctgg 3421cctcccctga gtacgtgaac ctccccatca atggcaacgg gaaacagtga gggttggggg 3481gactctgagc gggaggcaga gtttgccttc ctttctccag gaccaataaa agggctaaga 3541gagctactat gagcactgtg tttcctggga cggggcttag gggttctcag cctc

A KRAS-associated malignant tumor or KRAS-associated cancer is definedherein as (a) a cancer cell or tumor cell containing a somatic KRASmutation, or (b) a cancer cell or tumor cell with an abnormal expressionlevel of KRAS including, but not limited to, amplification of the KRASencoding DNA, or over-expression of the KRAS gene, or under-expressionof the KRAS gene when compared to level found in normal, non-cancercells.

Table 2 shows the amino acid sequence of the KRAS protein and identifiesthe mutations associated with cancer.

TABLE 2 Amino acid sequence of KRAS protein and mutations associatedwith cancer (SEQ ID NO: 2) KRAS protein coding sequence, Isoform 2A(identifier: P01116-1)        10         20         30         40         50 MTEYKLVVVGAGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET        60         70         80         90        100 CLLDILDTAGQEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI       110        120        130        140        150 KRVKDSEDVPMVLVGNKCDL PSRTVDTKQA QDLARSYGIP FIETSAKTRQ       160        170        180 RVEDAFYTLV REIRQYRLKK ISKEEKTPGCVKIKKCIIM Mutations at G → A in a colorectal cancer sample position 12:G → C in lung carcinoma G → D in pancreatic carcinoma, GASC and lungcarcinoma G → S in lung carcinoma and GASC G → V in lung carcinoma,pancreatic carcinoma, colon cancer and GASC Mutations at G → D in abreast carcinoma cell line and GASC position 13: G → R in pylocyticastrocytoma; amplification of the Ras pathway Mutations at Q → H in lungcarcinoma position 61: Q → R in a colorectal cancer

QIAGEN's THERASCREEN KRAS TEST is a genetic test designed to detect thepresence of seven mutations in the KRAS gene in colorectal cancer cells.

Therapeutic Compositions

After a subject is diagnosed as having a neoplasia, e.g., a lung canceror a pancreatic cancer, associated with a KRAS mutation or a KRASamplification, a method of treatment involving suppression of GST-π isselected.

In one embodiment, the inhibitory nucleic acid molecules of theinvention are administered systemically in dosages from about 1 to 100mg/kg, e.g., 1, 5, 10, 20, 25, 50, 75, or 100 mg/kg.

In further embodiments, the dosage can range from about 25 to 500mg/m²/day.

Examples of an agent that suppresses GST-π as used herein include a drugthat suppresses GST-π production and/or activity, and a drug thatpromotes GST-π degradation and/or inactivation. Examples of the drugthat suppresses GST-π production include an RNAi molecule, a ribozyme,an antisense nucleic acid, a DNA/RNA chimera polynucleotide for DNAencoding GST-π, or a vector expressing same.

GST-pi and RNAi Molecules

One of ordinary skill in the art would understand that a reportedsequence may change over time and to incorporate any changes needed inthe nucleic acid molecules herein accordingly.

Embodiments of this invention can provide compositions and methods forgene silencing of GST-pi expression using small nucleic acid molecules.Examples of nucleic acid molecules include molecules active in RNAinterference (RNAi molecules), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules. Such molecules are capable of mediating RNAinterference against GST-pi gene expression.

The composition and methods disclosed herein can also be used intreating various kinds of malignant tumors in a subject.

The nucleic acid molecules and methods of this invention may be used todown regulate the expression of genes that encode GST-pi.

The compositions and methods of this invention can include one or morenucleic acid molecules, which, independently or in combination, canmodulate or regulate the expression of GST-pi protein and/or genesencoding GST-pi proteins, proteins and/or genes encoding GST-piassociated with the maintenance and/or development of diseases,conditions or disorders associated with GST-pi, such as malignant tumor.

The compositions and methods of this invention are described withreference to exemplary sequences of GST-pi. A person of ordinary skillin the art would understand that various aspects and embodiments of theinvention are directed to any related GST-pi genes, sequences, orvariants, such as homolog genes and transcript variants, andpolymorphisms, including single nucleotide polymorphism (SNP) associatedwith any GST-pi genes.

In some embodiments, the compositions and methods of this invention canprovide a double-stranded short interfering nucleic acid (siRNA)molecule that downregulates the expression of a GST-pi gene, for examplehuman GST-pi.

A RNAi molecule of this invention can be targeted to GST-pi and anyhomologous sequences, for example, using complementary sequences or byincorporating non-canonical base pairs, for example, mismatches and/orwobble base pairs, that can provide additional target sequences.

In instances where mismatches are identified, non-canonical base pairs,for example, mismatches and/or wobble bases can be used to generatenucleic acid molecules that target more than one gene sequence.

For example, non-canonical base pairs such as UU and CC base pairs canbe used to generate nucleic acid molecules that are capable of targetingsequences for differing GST-pi targets that share sequence homology.Thus, a RNAi molecule can be targeted to a nucleotide sequence that isconserved between homologous genes, and a single RNAi molecule can beused to inhibit expression of more than one gene.

In some aspects, the compositions and methods of this invention includeRNAi molecules that are active against GST-pi mRNA, where the RNAimolecule includes a sequence complementary to any mRNA encoding a GST-pisequence.

In some embodiments, a RNAi molecule of this disclosure can haveactivity against GST-pi RNA, where the RNAi molecule includes a sequencecomplementary to an RNA having a variant GST-pi encoding sequence, forexample, a mutant GST-pi gene known in the art to be associated withmalignant tumor.

In further embodiments, a RNAi molecule of this invention can include anucleotide sequence that can mediate silencing of GST-pi geneexpression.

Examples of RNAi molecules of this invention targeted to GST-π mRNA areshown in Table 3.

TABLE 3 RNAi molecule sequences for GST-π SEQ SENSE STRAND SEQ ANTISENSESTRAND Ref ID (5′-->3′) ID (5′-->3′) ID Pos NO SEQ ID NOS: 3 to 67 NOSEQ ID NOS: 68 to 132 A1 652 3 UCCCAGAACCAGGGAGGCAtt 68UGCCUCCCUGGUUCUGGGAca A10 635 4 CUUUUGAGACCCUGCUGUCtt 69GACAGCAGGGUCUCAAAAGgc A11 649 5 CUGUCCCAGAACCAGGGAGtt 70CUCCCUGGUUCUGGGACAGca A12 650 6 UGUCCCAGAACCAGGGAGGtt 71CCUCCCUGGUUCUGGGACAgc A13 631 7 AAGCCUUUUGAGACCCUGCtt 72GCAGGGUCUCAAAAGGCUUca A14 638 8 UUGAGACCCUGCUGUCCCAtt 73UGGGACAGCAGGGUCUCAAaa A15 636 9 UUUUGAGACCCUGCUGUCCtt 74GGACAGCAGGGUCUCAAAAgg A16 640 10 GAGACCCUGCUGUCCCAGAtt 75UCUGGGACAGCAGGGUCUCaa A17 332 11 GCUGGAAGGAGGAGGUGGUtt 76ACCACCUCCUCCUUCCAGCtc A18 333 12 CUGGAAGGAGGAGGUGGUGtt 77CACCACCUCCUCCUUCCAGct A19 321 13 UCAGGGCCAGAGCUGGAAGtt 78CUUCCAGCUCUGGCCCUGAtc A2 639 14 UGAGACCCUGCUGUCCCAGtt 79CUGGGACAGCAGGGUCUCAaa A20 323 15 AGGGCCAGAGCUGGAAGGAtt 80UCCUUCCAGCUCUGGCCCUga A21 331 16 AGCUGGAAGGAGGAGGUGGtt 81CCACCUCCUCCUUCCAGCUct A22 641 17 AGACCCUGCUGUCCCAGAAtt 82UUCUGGGACAGCAGGGUCUca A23 330 18 GAGCUGGAAGGAGGAGGUGtt 83CACCUCCUCCUUCCAGCUCtg A25 647 19 UGCUGUCCCAGAACCAGGGtt 84CCCUGGUUCUGGGACAGCAgg A26 653 20 CCCAGAACCAGGGAGGCAAtt 85UUGCCUCCCUGGUUCUGGGac A3 654 21 CCAGAACCAGGGAGGCAAGtt 86CUUGCCUCCCUGGUUCUGGga A4 637 22 UUUGAGACCCUGCUGUCCCtt 87GGGACAGCAGGGUCUCAAAag A5 642 23 GACCCUGCUGUCCCAGAACtt 88GUUCUGGGACAGCAGGGUCtc A6 319 24 GAUCAGGGCCAGAGCUGGAtt 89UCCAGCUCUGGCCCUGAUCtg A7 632 25 AGCCUUUUGAGACCCUGCUtt 90AGCAGGGUCUCAAAAGGCUtc A8 633 26 GCCUUUUGAGACCCUGCUGtt 91CAGCAGGGUCUCAAAAGGCtt A9 634 27 CCUUUUGAGACCCUGCUGUtt 92ACAGCAGGGUCUCAAAAGGct AG7 632 28 CGCCUUUUGAGACCCUGCAtt 93UGCAGGGUCUCAAAAGGCGtc AK1 257 29 CCUACACCGUGGUCUAUUUtt 94AAAUAGACCACGGUGUAGGgc AK10 681 30 UGUGGGAGACCAGAUCUCCtt 95GGAGAUCUGGUCUCCCACAat AK11 901 31 GCGGGAGGCAGAGUUUGCCtt 96GGCAAACUCUGCCUCCCGCtc AK12 922 32 CCUUUCUCCAGGACCAAUAtt 97UAUUGGUCCUGGAGAAAGGaa AK13/ 643 33 ACCCUGCUGUCCCAGAACCtt 98GGUUCUGGGACAGCAGGGUct A24 AK2 267 34 GGUCUAUUUCCCAGUUCGAtt 99UCGAACUGGGAAAUAGACCac AK3 512 35 CCCUGGUGGACAUGGUGAAtt 100UUCACCAUGUCCACCAGGGct AK4 560 36 ACAUCUCCCUCAUCUACACtt 101GUGUAGAUGAGGGAGAUGUat AK5 593 37 GCAAGGAUGACUAUGUGAAtt 102UUCACAUAGUCAUCCUUGCcc AK6 698 38 CCUUCGCUGACUACAACCUtt 103AGGUUGUAGUCAGCGAAGGag AK7 313 39 CUGGCAGAUCAGGGCCAGAtt 104UCUGGCCCUGAUCUGCCAGca AK8 421 40 GACGGAGACCUCACCCUGUtt 105ACAGGGUGAGGUCUCCGUCct AK9 590 41 CGGGCAAGGAUGACUAUGUtt 106ACAUAGUCAUCCUUGCCCGcc AU10 635 42 CUUUUGAGACCCUGCUGUAtt 107UACAGCAGGGUCUCAAAAGgc AU23 330 43 GAGCUGGAAGGAGGAGGUAtt 108UACCUCCUCCUUCCAGCUCtg AU24 643 44 ACCCUGCUGUCCCAGAACAtt 109UGUUCUGGGACAGCAGGGUct AU25 648 45 UGCUGUCCCAGAACCAGGAtt 110UCCUGGUUCUGGGACAGCAgg AU7 632 46 AGCCUUUUGAGACCCUGCAtt 111UGCAGGGUCUCAAAAGGCUtc AU9 634 47 CCUUUUGAGACCCUGCUGAtt 112UCAGCAGGGUCUCAAAAGGct B1 629 48 UGAAGCCUUUUGAGACCCUtt 113AGGGUCUCAAAAGGCUUCAgt B10 627 49 ACUGAAGCCUUUUGAGACCtt 114GGUCUCAAAAGGCUUCAGUtg B11 595 50 AAGGAUGACUAUGUGAAGGtt 115CCUUCACAUAGUCAUCCUUgc B12 596 51 AGGAUGACUAUGUGAAGGCtt 116GCCUUCACAUAGUCAUCCUtg B13 597 52 GGAUGACUAUGUGAAGGCAtt 117UGCCUUCACAUAGUCAUCCtt B14 564 53 CUCCCUCAUCUACACCAACtt 118GUUGGUGUAGAUGAGGGAGat B2 630 54 GAAGCCUUUUGAGACCCUGtt 119CAGGGUCUCAAAAGGCUUCag B3 563 55 UCUCCCUCAUCUACACCAAtt 120UUGGUGUAGAUGAGGGAGAtg B4 567 56 CCUCAUCUACACCAACUAUtt 121AUAGUUGGUGUAGAUGAGGga B5 566 57 CCCUCAUCUACACCAACUAtt 122UAGUUGGUGUAGAUGAGGGag B6 625 58 CAACUGAAGCCUUUUGAGAtt 123UCUCAAAAGGCUUCAGUUGcc B7 626 59 AACUGAAGCCUUUUGAGACtt 124GUCUCAAAAGGCUUCAGUUgc B8 628 60 CUGAAGCCUUUUGAGACCCtt 125GGGUCUCAAAAGGCUUCAGtt B9 565 61 UCCCUCAUCUACACCAACUtt 126AGUUGGUGUAGAUGAGGGAga BG3 563 62 GCUCCCUCAUCUACACCAAtt 127UUGGUGUAGAUGAGGGAGCtg BU2 630 63 GAAGCCUUUUGAGACCCUAtt 128UAGGGUCUCAAAAGGCUUCag BU10 627 64 ACUGAAGCCUUUUGAGACAtt 129UGUCUCAAAAGGCUUCAGUtg BU14 565 65 CUCCCUCAUCUACACCAAAtt 130UUUGGUGUAGAUGAGGGAGat BU4 567 66 CCUCAUCUACACCAACUAAtt 131UUAGUUGGUGUAGAUGAGGga C1- 934 67 ACCAAUAAAAUUUCUAAGAtt 132UCUUAGAAAUUUUAUUGGUcc 934 Key for Table 3: Upper case A, G, C and Urefer to ribo-A, ribo-G, ribo-C and ribo-U, respectively. The lower caseletters a, u, g, c, t refer to 2′-deoxy-A, 2′-deoxy-U, 2′-deoxy-G,2′-deoxy-C, and deoxythymidine respectively.

Examples of RNAi molecules of this invention targeted to GST-π mRNA areshown in Table 4.

TABLE 4 RNAi molecule sequences for GST-π SEQ SENSE STRAND SEQ ANTISENSESTRAND ID (5′-->3′) ID (5′-->3′) ID NO SEQ ID NOS: 133 to 158 NO SEQ IDNOS: 159 to 184 BU2′ 133 GAAGCCUUUUGAGACCCUANN 159 UAGGGUCUCAAAAGGCUUCNN14 134 GAAGCCUUUUGAGACCCUAUU 160 UAGGGUCUCAAAAGGCUUCUU 15 135GAAGCCUUUUGAGACCCUAUU 161 uagggucuCAAAAGGCUUCUU 16 136GAAGCCUUUUGAGACCCUAUU 162 UagggucuCAAAAGGCUUCUU 17 137GAAGCCUUUUGAGACCCUAUU 163 UAgggucuCAAAAGGCUUCUU 18 138GAAGCCUUUUGAGACCCUAUU 164 UAGggucuCAAAAGGCUUCUU 19 139GAAGCCUUUUGAGACCCUAUU 165 UAGGgucuCAAAAGGCUUCUU 20 140GAAGCCUUUUGAGACCCUAUU 166 uAgGgUcUCAAAAGGCUUCUU 21 141GAAGCCUUUUGAGACCCUAUU 167 UAgGgUcUCAAAAGGCUUCUU 22 142GAAGCCUUUUGAGACCCUAUU 168 UaGgGuCuCAAAAGGCUUCUU 23 143GAAGCCUUUUGAGACCCUAUU 169 UAGgGuCuCAAAAGGCUUCUU 24 144GAAGCCUUUUGAGACCCUAtt 170 UagggucuCAAAAGGCUUCUU 25 145GAAGCCUUUUGAGACCCUAUU 171 UAGGGUCUCAAAAGGCUUCUU 26 146GAAGCCUUUUGAGACCCUAUU 172 fUAGGGUCUCAAAAGGCUUCUU 27 147GAAGCCUUUUGAGACCCUAUU 173 uAGGGUCUCAAAAGGCUUCUU 28 148GAAGCCUUUUGAGACCCUAUU 174 UsAGGGUCUCAAAAGGCUUCUU 29 149GAAGCCUUUUGAGACCCUfAUU 175 fUAGGGUCUfCAAAAGGCfUUCUU 30 150GAAGCCUUUUGAGfACCCUfAUU 176 fUAGGGUCUfCAfAfAAGGCfUUCUU 31 151GAAGCCUUUUGAGACCCUAUU 177 UAGGGUCUCAAAAGGCUUCUU 31′ 152GAAGCCUUUUGAGACCCUAUU 178 fUAGGGUCUCAAAAGGCUUCUU 32 153GAAGCCUUUUGAGACCCUAUU 179 UAGGGUCUCAAAAGGCUUCUU 39 154GAAGCCUUUUGAGACCCUAUU 180 UAGgGuCuCAAAAGGCUUCUU 45 155GAAGCCUUUUGAGACCCUAUU 181 UAGgGuCuCAAAAGGCUUCUU 46 156GAAGCCUUUUGAGACCCUAUU 182 UAGgGuCuCAAAAGGCUUCUU 47 157GAAGCCUUUUGAGACCCUAUU 183 UAGgGuCuCAAAAGGCUUCUU 48 158GAAGCCUUUUGAGACCCUAUU 184 fUAGgGuCuCAAAAGGCUUCUU Key for Table 4: Uppercase A, G, C and U refer to ribo-A, ribo-G, ribo-C and ribo-U,respectively. The lower case letters a, u, g, c, t refer to 2′-deoxy-A,2′-deoxy-U, 2′-deoxy-G, 2′-deoxy-C, and deoxythymidine (dT = T = t)respectively. Underlining refers to 2′-OMe-substituted, e.g., U. Thelower case letter f refers to 2′-deoxy-2′-fluoro substitution, e.g. fUis 2′-deoxy-2′-fluoro-U. N is A, C, G, U, U, a, c, g, u, t, or amodified, inverted, or chemically modified nucleotide.

Examples of RNAi molecules of this invention targeted to GST-π mRNA areshown in Table 5.

TABLE 5 RNAi molecule sequences for GST-π SENSE STRAND ANTISENSE STRANDSEQ ID (5′-->3′) SEQ ID (5′-->3′) ID NO SEQ ID NOS: 185 to 196 NO SEQ IDNOS: 197 to 208 A9′ 185 CCUUUUGAGACCCUGCUGUNN 197 ACAGCAGGGUCUCAAAAGGNN1 186 CCUUUUGAGACCCUGCUGUUU 198 ACAGCAGGGUCUCAAAAGGUU 2 187CCUUUUGAGACCCUGCUGUUU 199 acagcaggGUCUCAAAAGGUU 3 188CCUUUUGAGACCCUGCUGUUU 200 AcagcaggGUCUCAAAAGGUU 4 189CCUUUUGAGACCCUGCUGUUU 201 ACagcaggGUCUCAAAAGGUU 5 190CCUUUUGAGACCCUGCUGUUU 202 ACAgcaggGUCUCAAAAGGUU 6 191CCUUUUGAGACCCUGCUGUUU 203 ACAGcaggGUCUCAAAAGGUU 7 192CCUUUUGAGACCCUGCUGUUU 204 aCaGcAgGGUCUCAAAAGGUU 8 193CCUUUUGAGACCCUGCUGUUU 205 ACaGcAgGGUCUCAAAAGGUU 9 194CCUUUUGAGACCCUGCUGUUU 206 AcAgCaGgGUCUCAAAAGGUU 10 195CCUUUUGAGACCCUGCUGUUU 207 ACAgCaGgGUCUCAAAAGGUU 11 196CCUUUUGAGACCCUGCUGUUU 208 AcagcaggGUCUCAAAAGGUU Key for Table 5: Uppercase A, G, C and U refer to ribo-A, ribo-G, ribo-C and ribo-U,respectively. The lower case letters a, u, g, c, t refer to 2′-deoxy-A,2′-deoxy-U, 2′-deoxy-G, 2′-deoxy-C, and deoxythymidine (dT = T = t)respectively. Underlining refers to 2′-OMe-substituted, e.g., U. Thelower case letter f refers to 2′-deoxy-2′-fluoro substitution, e.g. fUis 2′-deoxy-2′-fluoro-U. N is A, C, G, U, U, a, c, g, u, t, or amodified, inverted, or chemically modified nucleotide.

Examples of RNAi molecules of this invention targeted to GST-π mRNA areshown in Table 6.

TABLE 6 RNAi molecule sequences for GST-π SEQ SENSE STRAND SEQ ANTISENSESTRAND ID (5′-->3′) ID (5′-->3′) ID NO SEQ ID NOS: 209 to 223 NO SEQ IDNOS: 224 to 238 B13′ 209 GGAUGACUAUGUGAAGGCANN 224 UGCCUUCACAUAGUCAUCCNN4 210 GGAUGACUAUGUGAAGGCAUU 225 UGCCUUCACAUAGUCAUCCUU 5 211GGAUGACUAUGUGAAGGCAUU 226 ugccuucaCAUAGUCAUCCUU 6 212GGAUGACUAUGUGAAGGCAUU 227 UgccuucaCAUAGUCAUCCUU 7 213GGAUGACUAUGUGAAGGCAUU 228 UGccuucaCAUAGUCAUCCUU 8 214GGAUGACUAUGUGAAGGCAUU 229 UGCcuucaCAUAGUCAUCCUU 9 215GGAUGACUAUGUGAAGGCAUU 230 UGCCuucaCAUAGUCAUCCUU 10 216GGAUGACUAUGUGAAGGCAUU 231 uGcCuUcACAUAGUCAUCCUU 11 217GGAUGACUAUGUGAAGGCAUU 232 UGcCuUcACAUAGUCAUCCUU 12 218GGAUGACUAUGUGAAGGCAUU 233 UgCcUuCaCAUAGUCAUCCUU 13 219GGAUGACUAUGUGAAGGCAUU 234 UGCcUuCaCAUAGUCAUCCUU 14 220GGAUGACUAUGUGAAGGCAUU 235 UgccuucaCAUAGUCAUCCUU 15 221GGAUGACUAUfGUfGAAGGCAUU 236 UGCfCUUCACAUAGUCAUCCUU 17 222GGAUGACUAUGUGAAGGCAUU 237 UGCCUUCACAUAGUCAUCCUU 18 223GGAUGACUAUGUGAAGGCAUU 238 UGCCUUCACAUAGUCAUCCUU Key for Table 6: Uppercase A, G, C and U refer to ribo-A, ribo-G, ribo-C and ribo-U,respectively. The lower case letters a, u, g, c, t refer to 2′-deoxy-A,2′-deoxy-U, 2′-deoxy-G, 2′-deoxy-C, and deoxythymidine (dT = T = t)respectively. Underlining refers to 2′-OMe-substituted, e.g., U. Thelower case letter f refers to 2′-deoxy-2′-fluoro substitution, e.g. fUis 2′-deoxy-2′-fluoro-U. N is A, C, G, U, U, a, c, g, u, t, or amodified, inverted, or chemically modified nucleotide.

Examples of RNAi molecules of this invention targeted to GST-π mRNA areshown in Table 7.

TABLE 7 RNAi molecule sequences for GST-π SENSE STRAND ANTISENSE STRANDSEQ ID (5′-->3′) SEQ ID (5′-->3′) ID NO SEQ ID NOS: 239 to 250 NO SEQ IDNOS: 251 to 262 B2′ 239 GAAGCCUUUUGAGACCCUGNN 251 CAGGGUCUCAAAAGGCUUCNN1 240 GAAGCCUUUUGAGACCCUGUU 252 CAGGGUCUCAAAAGGCUUCUU 2 241GAAGCCUUUUGAGACCCUGUU 253 cagggucuCAAAAGGCUUCUU 3 242GAAGCCUUUUGAGACCCUGUU 254 CagggucuCAAAAGGCUUCUU 4 243GAAGCCUUUUGAGACCCUGUU 255 CAgggucuCAAAAGGCUUCUU 5 244GAAGCCUUUUGAGACCCUGUU 256 CAGggucuCAAAAGGCUUCUU 6 245GAAGCCUUUUGAGACCCUGUU 257 CAGGgucuCAAAAGGCUUCUU 7 246GAAGCCUUUUGAGACCCUGUU 258 cAgGgUcUCAAAAGGCUUCUU 8 247GAAGCCUUUUGAGACCCUGUU 259 CAgGgUcUCAAAAGGCUUCUU 9 248GAAGCCUUUUGAGACCCUGUU 260 CaGgGuCuCAAAAGGCUUCUU 10 249GAAGCCUUUUGAGACCCUGUU 261 CAGgGuCuCAAAAGGCUUCUU 11 250GAAGCCUUUUGAGACCCUGUU 262 CagggucuCAAAAGGCUUCUU Key for Table 7: Uppercase A, G, C and U refer to ribo-A, ribo-G, ribo-C and ribo-U,respectively. The lower case letters a, u, g, c, t refer to 2′-deoxy-A,2′-deoxy-U, 2′-deoxy-G, 2′-deoxy-C, and deoxythymidine (dT = T = t)respectively. Underlining refers to 2′-OMe-substituted, e.g., U. Thelower case letter f refers to 2′-deoxy-2′-fluoro substitution, e.g. fUis 2′-deoxy-2′-fluoro-U. N is A, C, G, U, U, a, c, g, u, t, or amodified, inverted, or chemically modified nucleotide.

Examples of RNAi molecules of this invention targeted to GST-π mRNA areshown in Table 8.

TABLE 8 RNAi molecule sequences for GST-π SENSE STRAND ANTISENSE STRANDSEQ ID (5′-->3′) SEQ ID (5′-->3′) ID NO SEQ ID NOS: 263 to 274 NO SEQ IDNOS: 275 to 286 B4′ 263 CCUCAUCUACACCAACUAUNN 275 AUAGUUGGUGUAGAUGAGGNN1 264 CCUCAUCUACACCAACUAUUU 276 AUAGUUGGUGUAGAUGAGGUU 2 265CCUCAUCUACACCAACUAUUU 277 auaguuggUGUAGAUGAGGUU 3 266CCUCAUCUACACCAACUAUUU 278 AuaguuggUGUAGAUGAGGUU 4 267CCUCAUCUACACCAACUAUUU 279 AUaguuggUGUAGAUGAGGUU 5 268CCUCAUCUACACCAACUAUUU 280 AUAguuggUGUAGAUGAGGUU 6 269CCUCAUCUACACCAACUAUUU 281 AUAGuuggUGUAGAUGAGGUU 7 270CCUCAUCUACACCAACUAUUU 282 aUaGuUgGUGUAGAUGAGGUU 8 271CCUCAUCUACACCAACUAUUU 283 AUaGuUgGUGUAGAUGAGGUU 9 272CCUCAUCUACACCAACUAUUU 284 AuAgUuGgUGUAGAUGAGGUU 10 273CCUCAUCUACACCAACUAUUU 285 AUAgUuGgUGUAGAUGAGGUU 11 274CCUCAUCUACACCAACUAUUU 286 AuaguuggUGUAGAUGAGGUU Key for Table 8: Uppercase A, G, C and U refer to ribo-A, ribo-G, ribo-C and ribo-U,respectively. The lower case letters a, u, g, c, t refer to 2′-deoxy-A,2′-deoxy-U, 2′-deoxy-G, 2′-deoxy-C, and deoxythymidine (dT = T = t)respectively. Underlining refers to 2′-OMe-substituted, e.g., U. Thelower case letter f refers to 2′-deoxy-2′-fluoro substitution, e.g. fUis 2′-deoxy-2′-fluoro-U. N is A, C, G, U, U, a, c, g, u, t, or amodified, inverted, or chemically modified nucleotide.

As used herein, the RNAi molecule denotes any molecule that causes RNAinterference, including, but not limited to, a duplex RNA such as siRNA(small interfering RNA), miRNA (micro RNA), shRNA (short hairpin RNA),ddRNA (DNA-directed RNA), piRNA (Piwi-interacting RNA), or rasiRNA(repeat associated siRNA) and modified forms thereof. These RNAimolecules may be commercially available or may be designed and preparedbased on known sequence information, etc. The antisense nucleic acidincludes RNA, DNA, PNA, or a complex thereof. As used herein, theDNA/RNA chimera polynucleotide includes, but is not limited to, adouble-strand polynucleotide composed of DNA and RNA that inhibits theexpression of a target gene.

In one embodiment, the agents of this invention contain siRNA as atherapeutic agent. An siRNA molecule can have a length from about 10-50or more nucleotides. An siRNA molecule can have a length from about15-45 nucleotides. An siRNA molecule can have a length from about 19-40nucleotides. An siRNA molecule can have a length of from 19-23nucleotides. An siRNA molecule of this invention can mediate RNAiagainst a target mRNA. Commercially available design tools and kits,such as those available from Ambion, Inc. (Austin, Tex.), and theWhitehead Institute of Biomedical Research at MIT (Cambridge, Mass.)allow for the design and production of siRNA.

Methods for Modulating GST-pi and Treating Malignant Tumor

Embodiments of this invention can provide RNAi molecules that can beused to down regulate or inhibit the expression of GST-pi and/or GST-piproteins.

In some embodiments, a RNAi molecule of this invention can be used todown regulate or inhibit the expression of GST-pi and/or GST-pi proteinsarising from GST-pi haplotype polymorphisms that may be associated witha disease or condition such as malignant tumor.

Monitoring of GST-pi protein or mRNA levels can be used to characterizegene silencing, and to determine the efficacy of compounds andcompositions of this invention.

The RNAi molecules of this disclosure can be used individually, or incombination with other siRNAs for modulating the expression of one ormore genes.

The RNAi molecules of this disclosure can be used individually, or incombination, or in conjunction with other known drugs for preventing ortreating diseases, or ameliorating symptoms of conditions or disordersassociated with GST-pi, including malignant tumor.

The RNAi molecules of this invention can be used to modulate or inhibitthe expression of GST-pi in a sequence-specific manner.

The RNAi molecules of this disclosure can include a guide strand forwhich a series of contiguous nucleotides are at least partiallycomplementary to a GST-pi mRNA.

In certain aspects, malignant tumor may be treated by RNA interferenceusing a RNAi molecule of this invention.

Treatment of malignant tumor may be characterized in suitable cell-basedmodels, as well as ex vivo or in vivo animal models.

Treatment of malignant tumor may be characterized by determining thelevel of GST-pi mRNA or the level of GST-pi protein in cells of affectedtissue.

Treatment of malignant tumor may be characterized by non-invasivemedical scanning of an affected organ or tissue.

Embodiments of this invention may include methods for preventing,treating, or ameliorating the symptoms of a GST-pi associated disease orcondition in a subject in need thereof.

In some embodiments, methods for preventing, treating, or amelioratingthe symptoms of malignant tumor in a subject can include administeringto the subject a RNAi molecule of this invention to modulate theexpression of a GST-pi gene in the subject or organism.

In some embodiments, this invention contemplates methods for downregulating the expression of a GST-pi gene in a cell or organism, bycontacting the cell or organism with a RNAi molecule of this invention.

GST-π inhibitory nucleic acid molecules can be nucleotide oligomers thatmay be employed as single-stranded or double-stranded nucleic acidmolecule to decrease GST-π expression. In one approach, the GST-πinhibitory nucleic acid molecule is a double-stranded RNA used for RNAinterference (RNAi)-mediated knockdown of GST-π gene expression. In oneembodiment, a double-stranded RNA (dsRNA) molecule is made that includesfrom eight to twenty-five (e.g., 8, 10, 12, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25) consecutive nucleotides of a nucleotide oligomer of theinvention. The dsRNA can be two complementary strands of RNA that haveduplexed, or a single RNA strand that has self-duplexed (small hairpin(sh)RNA).

In some embodiments, dsRNAs are about 21 or 22 base pairs, but may beshorter or longer, up to about 29 nucleotides. Double stranded RNA canbe made using standard techniques, e.g., chemical synthesis or in vitrotranscription. Kits are available, for example, from Ambion (Austin,Tex.) and Epicentre (Madison, Wis.).

Methods for expressing dsRNA in mammalian cells are described inBrummelkamp et al. Science 296:550-553, 2002; Paddison et al. Genes &Devel. 16:948-958, 2002; Paul et al. Nature Biotechnol. 20:505-508,2002; Sui et al., Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu etal. Proc. Natl. Acad. Sci. USA 99:6047-6052, 2002; Miyagishi et al.,Nature Biotechnol. 20:497-500, 2002; and Lee et al., Nature Biotechnol.20:500-505 2002, each of which is hereby incorporated by reference.

An inhibitory nucleic acid molecule that “corresponds” to a GST-π genecomprises at least a fragment of the double-stranded gene, such thateach strand of the double-stranded inhibitory nucleic acid molecule iscapable of binding to the complementary strand of the target GST-π gene.The inhibitory nucleic acid molecule need not have perfectcorrespondence to the reference GST-π sequence.

In one embodiment, a siRNA has at least about 85%, 90%, 95%, 96%, 97%,98%, or even 99% sequence identity with the target nucleic acid. Forexample, a 19 base pair duplex having 1-2 base pair mismatch isconsidered useful in the methods of the invention. In other embodiments,the nucleotide sequence of the inhibitory nucleic acid molecule exhibits1, 2, 3, 4, 5 or more mismatches.

The inhibitory nucleic acid molecules provided by the invention are notlimited to siRNAs, but include any nucleic acid molecule sufficient todecrease the expression of a GST-π nucleic acid molecule or polypeptide.Each of the DNA sequences provided herein may be used, for example, inthe discovery and development of therapeutic antisense nucleic acidmolecule to decrease the expression of GST-π. The invention furtherprovides catalytic RNA molecules or ribozymes. Such catalytic RNAmolecules can be used to inhibit expression of an GST-π nucleic acidmolecule in vivo. The inclusion of ribozyme sequences within anantisense RNA confers RNA-cleaving activity upon the molecule, therebyincreasing the activity of the constructs. The design and use of targetRNA-specific ribozymes is described in Haseloff et al., Nature334:585-591. 1988, and US 2003/0003469 A1, each of which is incorporatedby reference.

In various embodiments of this invention, the catalytic nucleic acidmolecule is formed in a hammerhead or hairpin motif. Examples of suchhammerhead motifs are described by Rossi et al., Aids Research and HumanRetroviruses, 8:183, 1992. Example of hairpin motifs are described byHampel et al., Biochemistry, 28:4929, 1989, and Hampel et al., NucleicAcids Research, 18: 299, 1990. Those skilled in the art will recognizethat what is needed in an enzymatic nucleic acid molecule is a specificsubstrate binding site that is complementary to one or more of thetarget gene RNA regions, and that it have nucleotide sequences within orsurrounding that substrate binding site which impart an RNA cleavingactivity to the molecule.

Table 9 shows the mRNA coding sequence of GST-π.

TABLE 9 Glutathione S-transferase-π1 mRNA coding sequence, NCBIReference Sequence: NM_000852.3, GeneID: 2950, Hugo gene NomenclatureCommittee: HGNC: 4638, Human Protein Reference Database: HPRD: 00614(SEQ ID NO: 287) 1 tgggaaagag ggaaaggctt ccccggccag ctgcgcggcgactccgggga ctccagggcg 61 cccctctgcg gccgacgccc ggggtgcagc ggccgccggggctggggccg gcgggagtcc 121 gcgggaccct ccagaagagc ggccggcgcc gtgactcagcactggggcgg agcggggcgg 181 gaccaccctt ataaggctcg gaggccgcga ggccttcgctggagtttcgc cgccgcagtc 241 ttcgccacca tgccgcccta caccgtggtc tatttcccagttcgaggccg ctgcgcggcc 301 ctgcgcatgc tgctggcaga tcagggccag agctggaaggaggaggtggt gaccgtggag 361 acgtggcagg agggctcact caaagcctcc tgcctatacgggcagctccc caagttccag 421 gacggagacc tcaccctgta ccagtccaat accatcctgcgtcacctggg ccgcaccctt 481 gggctctatg ggaaggacca gcaggaggca gccctggtggacatggtgaa tgacggcgtg 541 gaggacctcc gctgcaaata catctccctc atctacaccaactatgaggc gggcaaggat 601 gactatgtga aggcactgcc cgggcaactg aagccttttgagaccctgct gtcccagaac 661 cagggaggca agaccttcat tgtgggagac cagatctccttcgctgacta caacctgctg 721 gacttgctgc tgatccatga ggtcctagcc cctggctgcctggatgcgtt ccccctgctc 781 tcagcatatg tggggcgcct cagtgcccgg cccaagctcaaggccttcct ggcctcccct 841 gagtacgtga acctccccat caatggcaac gggaaacagtgagggttggg gggactctga 901 gcgggaggca gagtttgcct tcctttctcc aggaccaataaaatttctaa gagagctaaa 961 aaaaaaaaaa aaaaaaaaaa aaaaaa

The drug that suppresses GST-π production or activity can be an RNAimolecule, a ribozyme, an antisense nucleic acid, a DNA/RNA chimerapolynucleotide for DNA encoding GST-π, or a vector expressing same, interms of high specificity and a low possibility of side effects.

Suppression of GST-π may be determined by the expression or activity ofGST-π in cells being suppressed compared with a case in which a GST-πsuppressing agent is not utilized. Expression of GST-π may be evaluatedby any known technique; examples thereof include an immunoprecipitationmethod utilizing an anti-GST-π antibody, EIA, ELISA, IRA, IRMA, awestern blot method, an immunohistochemical method, animmunocytochemical method, a flow cytometry method, varioushybridization methods utilizing a nucleic acid that specificallyhybridizes with a nucleic acid encoding GST-π or a unique fragmentthereof, or a transcription product (e.g., mRNA) or splicing product ofsaid nucleic acid, a northern blot method, a Southern blot method, andvarious PCR methods.

The activity of GST-π may be evaluated by analyzing a known activity ofGST-π including binding to a protein such as, for example, Raf-1 (inparticular phosphorylated Raf-1) or EGFR (in particular phosphorylatedEGFR) by means of any known method such as for example animmunoprecipitation method, a western blot method, amass analysismethod, a pull-down method, or a surface plasmon resonance (SPR) method.

Whether or not GST-π is being expressed in certain cells may bedetermined by detecting expression of GST-π in cells. Expression ofGST-π may be detected by any technique known in the art.

Examples of the mutated KRAS include, but are not limited to, thosehaving a mutation that causes constant activation of KRAS, such as amutation that inhibits endogenous GTPase or a mutation that increasesthe guanine nucleotide exchange rate. Specific examples of such mutationinclude, but are not limited to, for example, mutation in amino acids12, 13 and/or 61 in human KRAS (inhibiting endogenous GTPase) andmutation in amino acids 116 and/or 119 in human KRAS (increasing guaninenucleotide exchange rate) (Bos, Cancer Res. 1989; 49 (17): 4682-9, Leviet al., Cancer Res. 1991; 51 (13): 3497-502).

In some embodiments of the present invention, the mutated KRAS can be aKRAS having a mutation in at least one of amino acids 12, 13, 61, 116,and 119 of human KRAS. In one embodiment of the present invention, themutated KRAS has a mutation at amino acid 12 of human KRAS. In someembodiments, the mutated KRAS may be one that induces overexpression ofGST-π. Cells having mutated KRAS may exhibit overexpression of GST-π.

Detection of mutated KRAS may be carried out using any known technique,e.g., selective hybridization by means of a nucleic acid probe specificto a known mutation sequence, an enzyme mismatch cleavage method,sequencing (Bos, Cancer Res. 1989; 49 (17): 4682-9), and a PCR-RFLPmethod (Miyanishi et al., Gastroenterology. 2001; 121 (4): 865-74).).

Detection of GST-π expression may be carried out using any knowntechnique. Whether or not GST-π is being overexpressed may be evaluatedby for example comparing the degree of expression of GST-π in cellshaving mutated KRAS with the degree of expression of GST-π in the sametype of cells having normal KRAS. In this situation, GST-π is beingoverexpressed if the degree of expression of GST-π in cells havingmutated KRAS exceeds the degree of expression of GST-π in the same typeof cells having normal KRAS.

In one aspect, the invention features a vector encoding an inhibitorynucleic acid molecule of any of the above aspects. In a particularembodiment, the vector is a retroviral, adenoviral, adeno-associatedviral, or lentiviral vector. In another embodiment, the vector containsa promoter suitable for expression in a mammalian cell.

The amount of active RNA interference inducing ingredient formulated inthe composition of the present invention may be an amount that does notcause an adverse effect exceeding the benefit of administration. Such anamount may be determined by an in vitro test using cultured cells, or atest in a model animal such as a mouse, a rat, a dog, or a pig, etc.,and such test methods are well known to a person skilled in the art.

The amount of active ingredient formulated can vary according to themanner in which the agent or composition is administered. For example,when a plurality of units of the composition is used for oneadministration, the amount of active ingredient to be formulated in oneunit of the composition may be determined by dividing the amount ofactive ingredient necessary for one administration by said plurality ofunits.

This invention also relates to a process for producing an agent orcomposition for suppressing GST-π, and the use of a drug that suppressesGST-π in the production of an agent or composition for reducing orshrinking malignant tumors.

RNA Interference

RNA interference (RNAi) refers to sequence-specific post-transcriptionalgene silencing in animals mediated by short interfering RNAs (siRNAs).See, e.g., Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Fire et al.,Nature, 1998, Vol. 391, pp. 806811; Sharp, Genes & Development, 1999,Vol. 13, pp. 139-141.

An RNAi response in cells can be triggered by a double stranded RNA(dsRNA), although the mechanism is not yet fully understood. CertaindsRNAs in cells can undergo the action of Dicer enzyme, a ribonucleaseIII enzyme. See, e.g., Zamore et al., Cell, 2000, Vol. 101, pp. 25-33;Hammond et al., Nature, 2000, Vol. 404, pp. 293-296. Dicer can processthe dsRNA into shorter pieces of dsRNA, which are siRNAs.

In general, siRNAs can be from about 21 to about 23 nucleotides inlength and include a base pair duplex region about 19 nucleotides inlength.

RNAi involves an endonuclease complex known as the RNA induced silencingcomplex (RISC). An siRNA has an antisense or guide strand which entersthe RISC complex and mediates cleavage of a single stranded RNA targethaving a sequence complementary to the antisense strand of the siRNAduplex. The other strand of the siRNA is the passenger strand. Cleavageof the target RNA takes place in the middle of the region complementaryto the antisense strand of the siRNA duplex See, e.g., Elbashir et al.,Genes & Development, 2001, Vol. 15, pp. 188-200.

As used herein, the term “sense strand” refers to a nucleotide sequenceof a siRNA molecule that is partially or fully complementary to at leasta portion of a corresponding antisense strand of the siRNA molecule. Thesense strand of a siRNA molecule can include a nucleic acid sequencehaving homology with a target nucleic acid sequence.

As used herein, the term “antisense strand” refers to a nucleotidesequence of a siRNA molecule that is partially or fully complementary toat least a portion of a target nucleic acid sequence. The antisensestrand of a siRNA molecule can include a nucleic acid sequence that iscomplementary to at least a portion of a corresponding sense strand ofthe siRNA molecule.

RNAi molecules can down regulate or knock down gene expression bymediating RNA interference in a sequence-specific manner. See, e.g.,Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Elbashir et al., Nature,2001, Vol. 411, pp. 494-498; Kreutzer et al., WO2000/044895;Zernicka-Goetz et al., WO2001/36646; Fire et al., WO1999/032619;Plaetinck et al., WO2000/01846; Mello et al., WO2001/029058.

As used herein, the terms “inhibit,” “down-regulate,” or “reduce” withrespect to gene expression means that the expression of the gene, or thelevel of mRNA molecules encoding one or more proteins, or the activityof one or more of the encoded proteins is reduced below that observed inthe absence of a RNAi molecule or siRNA of this invention. For example,the level of expression, level of mRNA, or level of encoded proteinactivity may be reduced by at least 1%, or at least 10%, or at least20%, or at least 50%, or at least 90%, or more from that observed in theabsence of a RNAi molecule or siRNA of this invention.

RNAi molecules can also be used to knock down viral gene expression, andtherefore affect viral replication.

RNAi molecules can be made from separate polynucleotide strands: a sensestrand or passenger strand, and an antisense strand or guide strand. Theguide and passenger strands are at least partially complementary. Theguide strand and passenger strand can form a duplex region having fromabout 15 to about 49 base pairs.

In some embodiments, the duplex region of a siRNA can have 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 base pairs.

In certain embodiments, a RNAi molecule can be active in a RISC complex,with a length of duplex region active for RISC.

In additional embodiments, a RNAi molecule can be active as a Dicersubstrate, to be converted to a RNAi molecule that can be active in aRISC complex.

In some aspects, a RNAi molecule can have complementary guide andpassenger sequence portions at opposing ends of a long molecule, so thatthe molecule can form a duplex region with the complementary sequenceportions, and the strands are linked at one end of the duplex region byeither nucleotide or non-nucleotide linkers. For example, a hairpinarrangement, or a stem and loop arrangement. The linker interactionswith the strands can be covalent bonds or non-covalent interactions.

A RNAi molecule of this disclosure may include a nucleotide,non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins thesense region of the nucleic acid to the antisense region of the nucleicacid. A nucleotide linker can be a linker of 2 nucleotides in length,for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Thenucleotide linker can be a nucleic acid aptamer. By “aptamer” or“nucleic acid aptamer” as used herein refers to a nucleic acid moleculethat binds specifically to a target molecule wherein the nucleic acidmolecule has sequence that includes a sequence recognized by the targetmolecule in its natural setting. Alternately, an aptamer can be anucleic acid molecule that binds to a target molecule, where the targetmolecule does not naturally bind to a nucleic acid. For example, theaptamer can be used to bind to a ligand-binding domain of a protein,thereby preventing interaction of the naturally occurring ligand withthe protein. See, e.g., Gold et al., Annu Rev Biochem, 1995, Vol. 64,pp. 763-797; Brody et al., J. Biotechnol., 2000, Vol. 74, pp. 5-13;Hermann et al., Science, 2000, Vol. 287, pp. 820-825.

Examples of a non-nucleotide linker include an abasic nucleotide,polyether, polyamine, polyamide, peptide, carbohydrate, lipid,polyhydrocarbon, or other polymeric compounds, for example polyethyleneglycols such as those having from 2 to 100 ethylene glycol units. Someexamples are described in Seela et al., Nucleic Acids Research, 1987,Vol. 15, pp. 3113-3129; Cload et al., J. Am. Chem. Soc., 1991, Vol. 113,pp. 6324-6326; Jaeschke et al., Tetrahedron Lett., 1993, Vol. 34, pp.301; Arnold et al., WO1989/002439; Usman et al., WO1995/006731; Dudyczet al., WO1995/011910, and Ferentz et al., J. Am. Chem. Soc., 1991, Vol.113, pp. 4000-4002.

A RNAi molecule can have one or more overhangs from the duplex region.The overhangs, which are non-base-paired, single strand regions, can befrom one to eight nucleotides in length, or longer. An overhang can be a3′-end overhang, wherein the 3′-end of a strand has a single strandregion of from one to eight nucleotides. An overhang can be a 5′-endoverhang, wherein the 5′-end of a strand has a single strand region offrom one to eight nucleotides.

The overhangs of a RNAi molecule can have the same length, or can bedifferent lengths.

A RNAi molecule can have one or more blunt ends, in which the duplexregion ends with no overhang, and the strands are base paired to the endof the duplex region.

A RNAi molecule of this disclosure can have one or more blunt ends, orcan have one or more overhangs, or can have a combination of a blunt endand an overhang end.

A 5′-end of a strand of a RNAi molecule may be in a blunt end, or can bein an overhang. A 3′-end of a strand of a RNAi molecule may be in ablunt end, or can be in an overhang.

A 5′-end of a strand of a RNAi molecule may be in a blunt end, while the3′-end is in an overhang. A 3′-end of a strand of a RNAi molecule may bein a blunt end, while the 5′-end is in an overhang.

In some embodiments, both ends of a RNAi molecule are blunt ends.

In additional embodiments, both ends of a RNAi molecule have anoverhang.

The overhangs at the 5′- and 3′-ends may be of different lengths.

In certain embodiments, a RNAi molecule may have a blunt end where the5′-end of the antisense strand and the 3′-end of the sense strand do nothave any overhanging nucleotides.

In further embodiments, a RNAi molecule may have a blunt end where the3′-end of the antisense strand and the 5′-end of the sense strand do nothave any overhanging nucleotides.

A RNAi molecule may have mismatches in base pairing in the duplexregion.

Any nucleotide in an overhang of a RNAi molecule can be adeoxyribonucleotide, or a ribonucleotide.

One or more deoxyribonucleotides may be at the 5′-end, where the 3′-endof the other strand of the RNAi molecule may not have an overhang, ormay not have a deoxyribonucleotide overhang.

One or more deoxyribonucleotides may be at the 3′-end, where the 5′-endof the other strand of the RNAi molecule may not have an overhang, ormay not have a deoxyribonucleotide overhang.

In some embodiments, one or more, or all of the overhang nucleotides ofa RNAi molecule may be 2′-deoxyribonucleotides.

Dicer Substrate RNAi Molecules

In some aspects, a RNAi molecule can be of a length suitable as a Dicersubstrate, which can be processed to produce a RISC active RNAimolecule. See, e.g., Rossi et al., US2005/0244858.

A Dicer substrate dsRNA can be of a length sufficient such that it isprocessed by Dicer to produce an active RNAi molecule, and may furtherinclude one or more of the following properties: (i) the Dicer substratedsRNA can be asymmetric, for example, having a 3′ overhang on theantisense strand, and (ii) the Dicer substrate dsRNA can have a modified3′ end on the sense strand to direct orientation of Dicer binding andprocessing of the dsRNA to an active RNAi molecule.

Methods of Use of RNAi Molecules

The nucleic acid molecules and RNAi molecules of this invention may bedelivered to a cell or tissue by direct application of the molecules, orwith the molecules combined with a carrier or a diluent.

The nucleic acid molecules and RNAi molecules of this invention can bedelivered or administered to a cell, tissue, organ, or subject by directapplication of the molecules with a carrier or diluent, or any otherdelivery vehicle that acts to assist, promote or facilitate entry into acell, for example, viral sequences, viral material, or lipid or liposomeformulations.

The nucleic acid molecules and RNAi molecules of this invention can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues. The nucleic acid or nucleic acidcomplexes can be locally administered to relevant tissues ex vivo, or invivo through direct dermal application, transdermal application, orinjection.

Delivery systems may include, for example, aqueous and nonaqueous gels,creams, emulsions, microemulsions, liposomes, ointments, aqueous andnonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders,and can contain excipients such as solubilizers and permeationenhancers.

A GST-π inhibitory nucleic acid molecule of this invention may beadministered within a pharmaceutically-acceptable diluents, carrier, orexcipient, in unit dosage form. Conventional pharmaceutical practice maybe employed to provide suitable formulations or compositions toadminister the compounds to patients suffering from a disease that iscaused by excessive cell proliferation. Administration may begin beforethe patient is symptomatic. Any appropriate route of administration maybe employed, for example, administration may be parenteral, intravenous,intraarterial, subcutaneous, intratumoral, intramuscular, intracranial,intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular,intrathecal, intracistemal, intraperitoneal, intranasal, aerosol,suppository, or oral administration. For example, therapeuticformulations may be in the form of liquid solutions or suspensions; fororal administration, formulations may be in the form of tablets orcapsules; and for intranasal formulations, in the form of powders, nasaldrops, or aerosols.

Compositions and methods of this disclosure can include an expressionvector that includes a nucleic acid sequence encoding at least one RNAimolecule of this invention in a manner that allows expression of thenucleic acid molecule.

The nucleic acid molecules and RNAi molecules of this invention can beexpressed from transcription units inserted into DNA or RNA vectors.Recombinant vectors can be DNA plasmids or viral vectors. Viral vectorscan be used that provide for transient expression of nucleic acidmolecules.

For example, the vector may contain sequences encoding both strands of aRNAi molecule of a duplex, or a single nucleic acid molecule that isself-complementary and thus forms a RNAi molecule. An expression vectormay include a nucleic acid sequence encoding two or more nucleic acidmolecules.

A nucleic acid molecule may be expressed within cells from eukaryoticpromoters. Those skilled in the art realize that any nucleic acid can beexpressed in eukaryotic cells from the appropriate DNA/RNA vector.

In some aspects, a viral construct can be used to introduce anexpression construct into a cell, for transcription of a dsRNA constructencoded by the expression construct.

Lipid formulations can be administered to animals by intravenous,intramuscular, or intraperitoneal injection, or orally or by inhalationor other methods as are known in the art.

Pharmaceutically acceptable formulations for administeringoligonucleotides are known and can be used.

In one embodiment of the above method, the inhibitory nucleic acidmolecule is administered at a dosage of about 5 to 500 mg/m²/day, e.g.,5, 25, 50, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/m²/day.

Methods known in the art for making formulations are found, for example,in “Remington: The Science and Practice of Pharmacy” Ed. A. R. Gennaro,Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000.

Formulations for parenteral administration may, for example, containexcipients, sterile water, or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds. Otherpotentially useful parenteral delivery systems for GST-π inhibitorynucleic acid molecules include ethylene-vinyl acetate copolymerparticles, osmotic pumps, implantable infusion systems, and liposomes.Formulations for inhalation may contain excipients, for example,lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may beoily solutions for administration in the form of nasal drops, or as agel.

The formulations can be administered to human patients intherapeutically effective amounts (e.g., amounts which prevent,eliminate, or reduce a pathological condition) to provide therapy for aneoplastic disease or condition. The preferred dosage of a nucleotideoligomer of the invention can depend on such variables as the type andextent of the disorder, the overall health status of the particularpatient, the formulation of the compound excipients, and its route ofadministration.

All of the above methods for reducing malignant tumors may be either anin vitro method or an in vivo method. Dosage may be determined by an invitro test using cultured cells, etc., as is known in the art. Aneffective amount may be an amount that reduces tumor size in KRASassociated tumors by at least 10%, at least 20%, or at least 30%, or atleast 40%, or at least 50%, or at least 60%, or at least 70%, or atleast 80%, or at least 90%, up to 100% of the tumor size.

A pharmaceutical composition of this invention can be effective intreating a KRAS associated disease. Examples of the diseases include adisease due to abnormal cell proliferation, a disease due to KRASmutation, and a disease due to GST-π overexpression.

Examples of the disease due to abnormal cell proliferation includemalignant tumors, hyperplasia, keloid, Cushing's syndrome, primaryaldosteronism, erythroplakia, polycythemia vera, leukoplakia,hyperplastic scar, lichen planus, and lentiginosis.

Examples of the disease due to KRAS mutation include malignant tumor(also called a cancer or a malignant neoplasm).

Examples of the disease due to GST-π overexpression include malignanttumor.

Examples of cancer include sarcomas such as fibrosarcoma, malignantfibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma,angiosarcoma, Kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma,chondrosarcoma, and osteosarcoma, carcinomas such as brain tumor, headand neck carcinoma, breast carcinoma, lung carcinoma, esophagealcarcinoma, gastric carcinoma, duodenal carcinoma, colon carcinoma,rectal carcinoma, liver carcinoma, pancreatic carcinoma, gall bladdercarcinoma, bile duct carcinoma, renal carcinoma, ureteral carcinoma,bladder carcinoma, prostate carcinoma, testicular carcinoma, uterinecarcinoma, ovarian carcinoma, skin carcinoma, leukemia, and malignantlymphoma.

Cancer includes epithelial malignancy and non-epithelial malignancy. Acancer can be present at any site of the body, for example, the brain,head and neck, chest, limbs, lung, heart, thymus, esophagus, stomach,small intestine (duodenum, jejunum, ileum), large intestine (colon,cecum, appendix, rectum), liver, pancreas, gallbladder, kidney, urinaryduct, bladder, prostate, testes, uterus, ovary, skin, striated muscle,smooth muscle, synovial membrane, cartilage, bone, thyroid, adrenalgland, peritoneum, mesentery, bone marrow, blood, vascular system,lymphatic system such as lymph node, lymphatic fluid, etc.

In one embodiment of the present invention, the cancer includes cancercells having the mutated KRAS defined above. In another embodiment, thecancer includes cancer cells that exhibit hormone- or growthfactor-independent proliferation. In further embodiments, a cancerincludes cancer cells exhibiting GST-π overexpression.

EXAMPLES Example 1

siRNAs of this invention targeted to GST-π were found to be active forgene silencing in vitro. The dose-dependent activities of GST-π siRNAsfor gene knockdown were found to exhibit an IC50 below about 250picomolar (pM), and as low as 1 pM.

In vitro transfection was performed in an A549 cell line to determinesiRNA knockdown efficacy. Dose dependent knockdown for GST-π mRNA wasobserved with siRNAs of Table 3, as shown in Table 10.

TABLE 10 Dose dependent knockdown for GST-π mRNA in an A549 cell linesiRNA structure IC50 (pM) A9 (SEQ ID NOs: 27 and 92) 24 B2 (SEQ ID NOs:54 and 119) 121 B3 (SEQ ID NOs: 55 and 120) 235 B4 (SEQ ID NOs: 56 and121) 229 B13 (SEQ ID NOs: 52 and 117) 17 BU2 (SEQ ID NOs: 63 and 128) 31

As shown in Table 10, the activities of GST-π siRNAs of Table 3 were inthe range 17-235 pM, which is suitable for many uses, including as adrug agent to be used in vivo.

Example 2

The structure of GST-π siRNAs of this invention having deoxynucleotideslocated in the seed region of the antisense strand of the siRNA providedunexpectedly and advantageously increased gene knockdown activity invitro.

In vitro transfection was performed in an A549 cell line to determineknockdown efficacy for GST-π siRNAs based on structure BU2′ (SEQ IDNOs:133 and 159). Dose dependent knockdown of GST-π mRNA was observedwith GST-π siRNAs based on structure BU2′ as shown in Table 11.

TABLE 11 Dose dependent knockdown of GST-π mRNA in an A549 cell line forGST-π siRNAs based on structure BU2′ GST-π siRNA structure IC50 (pM) BU2with no deoxynucleotides in the duplex region 31 (SEQ ID NOs: 63 and128) BU2 with deoxynucleotides in positions 3, 5, and 7 of 5 the seedregion antisense strand (SEQ ID NOs: 141 and 167) BU2 withdeoxynucleotides in positions 4, 6, and 8 of 8 the seed region antisensestrand (SEQ ID NOs: 143 and 169) BU2 with deoxynucleotides in positions4, 6, and 8 of 5 the seed region antisense strand (SEQ ID NOs: 158 and184)

As shown in Table 11, the activities of GST-π siRNAs based on structureBU2′ having three deoxynucleotides in the seed region of the antisensestrand were surprisingly and unexpectedly increased by up to 6-fold, ascompared to a GST-π siRNA without deoxynucleotides in the duplex region.

These data show that GST-π siRNAs having a structure with threedeoxynucleotides located at positions 3, 5 and 7, or at positions 4, 6and 8 in the seed region of the antisense strand provided surprisinglyincreased gene knockdown activity as compared to a GST-π siRNA withoutdeoxynucleotides in the duplex region.

The activities shown in Table 11 for GST-π siRNAs having threedeoxynucleotides in the seed region of the antisense strand were in therange 5 to 8 pM, which is exceptionally suitable for many uses,including as a drug agent to be used in vivo.

Example 3

The structure of GST-π siRNAs of this invention having deoxynucleotideslocated in the seed region of the antisense strand of the siRNA providedunexpectedly and advantageously increased gene knockdown activity invitro.

In vitro transfection was performed in an A549 cell line to determineknockdown efficacy for GST-π siRNAs based on structure A9′ (SEQ IDNOs:185 and 197). Dose dependent knockdown of GST-π mRNA was observedwith the GST-π siRNAs based on structure A9′, as shown in Table 12.

TABLE 12 Dose dependent knockdown of GST-π mRNA in an A549 cell line forGST-π siRNAs based on structure structure A9′ GST-π siRNA structure IC50(pM) A9 with no deoxynucleotides in the duplex region 24 (SEQ ID NOs: 27and 92) A9 with deoxynucleotides in positions 4, 6, and 8 of 1 the seedregion antisense strand (SEQ ID NOs: 195 and 207) A9 withdeoxynucleotides in positions 1, 3, 5, and 7 5 of the seed regionantisense strand (SEQ ID NOs: 192 and 204) A9 with deoxynucleotides inpositions 3-8 of the seed 6 region antisense strand (SEQ ID NOs: 189 and201) A9 with deoxynucleotides in positions 5-8 of the seed 7 regionantisense strand (SEQ ID NOs: 191 and 203) A9 with deoxynucleotides inpositions 3, 5, and 7 of 15 the seed region antisense strand (SEQ IDNOs: 193 and 205)

As shown in Table 12, the activities of GST-π siRNAs based on structureA9′ having three to six deoxynucleotides in the seed region of theantisense strand were surprisingly increased by up to 24-fold, ascompared to a GST-π siRNA without deoxynucleotides in the duplex region.

These data show that GST-π siRNAs having a structure with three to sixdeoxynucleotides located at positions 4, 6 and 8, or at positions 1, 3,5 and 7, or at positions 3-8, or at positions 5-8, or at positions 3, 5and 7 in the seed region of the antisense strand provided unexpectedlyincreased gene knockdown activity as compared to a GST-π siRNA withoutdeoxynucleotides in the duplex region.

The activity shown in Table 12 for GST-π siRNAs having three to sixdeoxynucleotides in the seed region of the antisense strand was in therange 1 to 15 pM, which is exceptionally suitable for many uses,including as a drug agent to be used in vivo.

Example 4

The structure of GST-π siRNAs having deoxynucleotides located in theseed region of the antisense strand of the siRNA provided unexpectedlyand advantageously increased gene knockdown activity in vitro.

In vitro transfection was performed in an A549 cell line to determineknockdown efficacy for GST-π siRNAs based on structure B13′ (SEQ IDNOs:209 and 224). Dose dependent knockdown of GST-π mRNA was observedwith the GST-π siRNAs based on structure B13′, as shown in Table 13.

TABLE 13 Dose dependent knockdown of GST-π mRNA in an A549 cell line forGST-π siRNAs based on structure B13′ GST-π siRNA structure IC50 (pM) B13with no deoxynucleotides in the duplex region 17 (SEQ ID NOs: 52 and117) B13 with deoxynucleotides in positions 4, 6, and 8 of 11 the seedregion antisense strand (SEQ ID NOs: 219 and 234)

As shown in Table 13, the activity of a GST-π siRNA based on structureB13′ having three deoxynucleotides in the seed region of the antisensestrand was unexpectedly increased, as compared to a GST-π siRNA withoutdeoxynucleotides in the duplex region.

These data show that GST-π siRNAs having a structure with threedeoxynucleotides located at positions 4, 6 and 8 in the seed region ofthe antisense strand provided unexpectedly increased gene knockdownactivity as compared to a GST-π siRNA without deoxynucleotides in theduplex region.

The activity shown in Table 13 for GST-π siRNAs having threedeoxynucleotides in the seed region of the antisense strand was in thepicomolar range at 11 pM, which is exceptionally suitable for many uses,including as a drug agent to be used in vivo.

Example 5

The structure of GST-π siRNAs having deoxynucleotides located in theseed region of the antisense strand of the siRNA provided unexpectedlyand advantageously increased gene knockdown activity in vitro.

In vitro transfection was performed in an A549 cell line to determineknockdown efficacy for GST-π siRNAs based on structure B4′ (SEQ IDNOs:263 and 275). Dose dependent knockdown of GST-π mRNA was observedwith the GST-π siRNAs based on structure B4′, as shown in Table 14.

TABLE 14 Dose dependent knockdown of GST-π mRNA in an A549 cell line forGST-π siRNAs based on structure B4′ GST-π siRNA structure IC50 (pM) B4with no deoxynucleotides in the duplex region 229 (SEQ ID NOs: 56 and121) B4 with deoxynucleotides in positions 3-8 of the seed 113 regionantisense strand (SEQ ID NOs: 267 and 279)

As shown in Table 14, the activities of GST-π siRNAs based on structureB4′ having six deoxynucleotides in the seed region of the antisensestrand were unexpectedly increased by more than two-fold, as compared toa GST-π siRNA without deoxynucleotides in the duplex region.

These data show that GST-π siRNAs having a structure with sixdeoxynucleotides located at positions 3-8 in the seed region of theantisense strand provided surprisingly increased gene knockdown activityas compared to a GST-π siRNA without deoxynucleotides in the duplexregion.

The activity shown in Table 14 for a GST-π siRNA having sixdeoxynucleotides in the seed region of the antisense strand was in thepicomolar range at 113 pM, which is exceptionally suitable for manyuses, including as a drug agent to be used in vivo.

Example 6

The structure of GST-π siRNAs having deoxynucleotides located in theseed region of the antisense strand of the siRNA provided unexpectedlyand advantageously increased gene knockdown activity in vitro.

In vitro transfection was performed in an A549 cell line to determineknockdown efficacy for GST-π siRNAs based on structure B2′ (SEQ IDNOs:239 and 251). Dose dependent knockdown of GST-π mRNA was observedwith the GST-π siRNAs based on structure B2′, as shown in Table 15.

TABLE 15 Dose dependent knockdown of GST-π mRNA in an A549 cell line forGST-π siRNAs based on structure B2′ GST-π siRNA structure IC50 (pM) B2with no deoxynucleotides in the duplex regioin 121 (SEQ ID NOs: 54 and119) B2 with deoxynucleotides in positions 5-8 of the seed 30 regionantisense strand (SEQ ID NOs: 245 and 257) B2 with deoxynucleotides inpositions 1, 3, 5, and 7 50 of the seed region antisense strand (SEQ IDNOs: 246 and 258) B2 with deoxynucleotides in positions 3, 5, and 7 of100 the seed region antisense strand (SEQ ID NOs: 246 and 259)

As shown in Table 15, the activities of GST-π siRNAs based on structureB2′ having three to four deoxynucleotides in the seed region of theantisense strand were surprisingly increased by up to 4-fold, ascompared to a GST-π siRNA without deoxynucleotides in the duplex region.

These data show that GST-π siRNAs having a structure with three to fourdeoxynucleotides located at positions 5-8, or at positions 1, 3, 5 and7, or at positions 3, 5 and 7 in the seed region of the antisense strandprovided unexpectedly increased gene knockdown activity as compared to aGST-π siRNA without deoxynucleotides in the duplex region.

The activities shown in Table 15 for GST-π siRNAs having three to fourdeoxynucleotides in the seed region of the antisense strand were in therange 30-100 pM, which is exceptionally suitable for many uses,including as a drug agent to be used in vivo.

Example 7

The structure of GST-π siRNAs containing one or more 2′-deoxy-2′-fluorosubstituted nucleotides provided unexpectedly increased gene knockdownactivity in vitro.

In vitro transfection was performed in an A549 cell line to determineknockdown efficacy for GST-π siRNAs based on structure BU2′ (SEQ IDNOs:133 and 159). Dose dependent knockdown of GST-π mRNA was observedwith the GST-π siRNAs based on structure BU2′, as shown in Table 16.

TABLE 16 Dose dependent knockdown of GST-π mRNA in an A549 cell line forGST-π siRNAs based on structure BU2′ GST-π siRNA structure IC50 (pM) BU2with no 2′-F deoxynucleotides 31 (SEQ ID NOs: 63 and 128) BU2 with seven2′-F deoxynucleotides, one in  3 position 1 at the 3′ end of theantisense strand (SEQ ID NOs: 150 and 176) BU2 with four 2′-Fdeoxynucleotides, one in position 11 1 at the 3′ end of the antisensestrand (SEQ ID NOs: 149 and 175) BU2 with one 2′-F deoxynucleotide inposition 1 at 13 the 3′ end of the antisense strand (SEQ ID NOs: 146 and172)

As shown in Table 16, the activities of GST-π siRNAs based on structureBU2′ having one or more 2′-F deoxynucleotides were surprisinglyincreased by up to 10-fold, as compared to a GST-π siRNA without 2′-Fdeoxynucleotides.

These data show that GST-π siRNAs having a structure with one or more2′-F deoxynucleotides provided unexpectedly increased gene knockdownactivity as compared to a GST-π siRNA without a 2′-F deoxynucleotide.

The activities shown in Table 16 for GST-π siRNAs having one or more2′-F deoxynucleotides were in the range 3 to 13 pM, which isexceptionally suitable for many uses, including as a drug agent to beused in vivo.

Example 8

The structure of GST-π siRNAs containing one or more 2′-deoxy-2′-fluorosubstituted nucleotides provided unexpectedly increased gene knockdownactivity in vitro.

In vitro transfection was performed in an A549 cell line to determineknockdown efficacy for GST-π siRNAs based on structure B13′ (SEQ IDNOs:209 and 224). Dose dependent knockdown of GST-π mRNA was observedwith the GST-π siRNAs based on structure B13′, as shown in Table 17.

TABLE 17 Dose dependent knockdown of GST-π mRNA in an A549 cell line forGST-π siRNAs based on structure B13′ GST-π siRNA structure IC50 (pM) B13with no 2′-F deoxynucleotides 17 (SEQ ID NOs: 52 and 117) B13 with three2′-F deoxynucleotides located in non- 6 overhang positions (SEQ ID NOs:221 and 236)

As shown in Table 17, the activity of a GST-π siRNA based on structureB13′ having three 2′-F deoxynucleotides located in non-overhangpositions was surprisingly increased by about 3-fold, as compared to aGST-π siRNA without 2′-F deoxynucleotides.

These data show that GST-π siRNAs having a structure with one or more2′-F deoxynucleotides provided unexpectedly increased gene knockdownactivity as compared to a GST-π siRNA without a 2′-F deoxynucleotide.

The activity shown in Table 17 for GST-π siRNAs having one or more 2′-Fdeoxynucleotides was in the picomolar range at 6 pM, which isexceptionally suitable for many uses, including as a drug agent to beused in vivo.

Example 9

Orthotopic A549 lung cancer mouse model. The GST-π siRNAs of thisinvention can exhibit profound reduction of orthotopic lung cancertumors in vivo. In this example, a GST-π siRNA provided gene knockdownpotency in vivo when administered in a liposomal formulation to theorthotopic lung cancer tumors in athymic nude mice.

In general, an orthotopic tumor model can exhibit direct clinicalrelevance for drug efficacy and potency, as well as improved predictiveability. In the orthotopic tumor model, tumor cells are implanteddirectly into the same kind of organ from which the cells originated.

The anti-tumor efficacy of the siRNA formulation against human lungcancer A549 was evaluated by comparing the final primary tumor weightsmeasured at necropsy for the treatment group and the vehicle controlgroup.

FIG. 1 shows orthotopic lung cancer tumor inhibition in vivo for a GST-πsiRNA based on structure BU2 (SEQ ID NOs:63 and 128). An orthotopic A549lung cancer mouse model was utilized with a relatively low dose at 2mg/kg of the siRNA targeted to GST-π.

The GST-π siRNA showed significant and unexpectedly advantageous lungtumor inhibition efficacy in this six-week study. As shown in FIG. 1,after 43 days, the GST-π siRNA showed markedly advantageous tumorinhibition efficacy, with final tumor average weights significantlyreduced by 2.8-fold as compared to control.

For this study, male NCr nu/nu mice, 5-6 weeks old, were used. Theexperimental animals were maintained in a HEPA filtered environmentduring the experimental period. The siRNA formulations were stored at 4°C. before use, and warmed to room temperature 10 minutes prior toinjection in mouse.

For this A549 human lung cancer orthotopic model, on the day of surgicalorthotopic implantation (SOI), the stock tumors were harvested from thesubcutaneous site of animals bearing A549 tumor xenograft and placed inRPMI-1640 medium. Necrotic tissues were removed and viable tissues werecut into 1.5-2 mm³ pieces. The animals were anesthetized with isofluraneinhalation and the surgical area was sterilized with iodine and alcohol.A transverse incision approximately 1.5 cm long was made in the leftchest wall of the mouse using a pair of surgical scissors. Anintercostal incision was made between the third and the fourth rib andthe left lung was exposed. One A549 tumor fragment was transplanted tothe surface of the lung with an 8-0 surgical suture (nylon). The chestwall was closed with a 6-0 surgical suture (silk). The lung wasre-inflated by intrathoracic puncture using a 3 cc syringe with a 25G×1½ needle to draw out the remaining air in the chest cavity. The chestwall was closed with a 6-0 surgical silk suture. All procedures of theoperation described above were performed with a 7× magnificationmicroscope under HEPA filtered laminar flow hoods.

Three days after tumor implantation, the model tumor-bearing mice wererandomly divided into groups of ten mice per group. For the group ofinterest, treatment of the ten mice was initiated three days after tumorimplantation.

For the group of interest, the formulation was (Ionizablelipid:cholesterol:DOPE:DOPC:DPPE-PEG-2K:D SPE-PEG-2K), a liposomalcomposition. The liposomes encapsulated the GST-π siRNA.

For the study endpoint, the experimental mice were sacrificed forty-twodays after treatment initiation. Primary tumors were excised and weighedon an electronic balance for subsequent analysis.

For an estimation of compound toxicity, the mean body weight of the micein the treated and control groups was maintained within the normal rangeduring the entire experimental period. Other symptoms of toxicity werenot observed in the mice.

Example 10

The GST-π siRNAs of this invention exhibited profound reduction ofcancer xenograft tumors in vivo. The GST-π siRNAs provided geneknockdown potency in vivo when administered in a liposomal formulationto the cancer xenograft tumors.

FIG. 2 shows tumor inhibition efficacy for a GST-π siRNA (SEQ ID Nos:158and 184). A cancer xenograft model was utilized with a relatively lowdose at 0.75 mg/kg of siRNA targeted to GST-π.

The GST-π siRNA showed significant and unexpectedly advantageous tumorinhibition efficacy within a few days after administration. After 36days, the GST-π siRNA showed markedly advantageous tumor inhibitionefficacy, with tumor volume reduced by 2-fold as compared to control.

As shown in FIG. 3, the GST-π siRNA demonstrated significant andunexpectedly advantageous tumor inhibition efficacy at the endpoint day.In particular, tumor weight was reduced by more than 2-fold.

The GST-π siRNA was administered in two injections (day 1 and 15) of aliposomal formulation having the composition (Ionizablelipid:Cholesterol:DOPE:DOPC:DPPE-PEG-2K) (25:30:20:20:5).

For the cancer xenograft model, an A549 cell line was obtained fromATCC. The cells were maintained in culture medium supplemented with 10%Fetal Bovine Serum and 100 U/ml penicillin and 100 μg/ml streptomycin.Cells were split 48 hrs before inoculation so that cells were in logphase growth when harvested. Cells were lightly trypsinized withtrypsin-EDTA and harvested from tissue culture. The number of viablecells was counted and determined in a hemocytometer in the presence oftrypan blue (only viable cells are counted). The cells were resuspendedto a concentration of 5×10⁷/ml in media without serum. Then the cellsuspension was mixed well with ice thawed BD matrigel at 1:1 ratio forinjection.

Mice were Charles River Laboratory Athymic Nude (nu/nu) Female Mice,immuno-compromised, 6-8 weeks old, 7-8 mice per group.

For tumor model preparation, each mouse was inoculated subcutaneously inthe right flank with 0.1 ml an inoculum of 2.5×10⁶ of A549 cells using a25 G needle and syringe, one inoculum per mouse. Mice were notanesthetized for inoculation.

For tumor volume measurements and randomization, tumor size was measuredto the nearest 0.1 mm. Tumor volumes were calculated using the formula:Tumor volume=length×width/2. Once the established tumors reachedapproximately 120-175 mm³, average tumor volume was about 150 mm³, themice were assigned into the various vehicle control and treatment groupssuch that the mean tumor volumes in the treated groups were within 10%of the mean tumor volume in the vehicle control group, ideally, the CV %of tumor volume was less than 25%. On the same day, test articles andcontrol vehicle were administered according to the dosing regimen. Tumorvolumes were monitored three times for week 1, twice for the rest ofweeks, including the day of study termination.

For dosage administration, on the dosing day, the test articles weretaken out from −80° C. freezer and thawed on ice. Before applied tosyringes, the bottle containing formulation was reverted by hands for afew times. All test articles were dosed at 0.75 mg/kg by IV, q2w×2, at10 ml/kg.

For body weight, mice were weighed to the nearest 0.1 g. Body weightswere monitored and recorded daily within 7 days post dosing for firstdose. Body weights were monitored and recorded twice for weeks, for therest of weeks, including the day of study termination.

For tumors collection, on 28 days post first dosing, tumor volume wasmeasured, and tumor was dissected for weight measurement, and stored forPD biomarker study. Tumor weight was recorded.

Example 11

The GST-π siRNAs of this invention demonstrated increased cancer celldeath by apoptosis of cancer cells in vitro. The GST-π siRNAs providedGST-π knockdown, which resulted in upregulation of PUMA, a biomarker forapoptosis and associated with loss in cell viability.

GST-π siRNA SEQ ID NOs:158 and 184, which contained a combination ofdeoxynucleotides in the seed region, a 2′-F substituted deoxynucleotide,and 2′-OMe substituted ribonucleotides, provided unexpectedly increasedapoptosis of cancer cells.

The level of expression of PUMA for GST-π siRNA SEQ ID NOs:158 and 184was measured as shown in FIG. 4. In FIG. 4, the expression of PUMA wasgreatly increased from 2-4 days after transfection of the GST-π siRNA.

These data show that the structure of GST-π siRNAs containing acombination of deoxynucleotides in the seed region, a 2′-F substituteddeoxynucleotide, and 2′-OMe substituted ribonucleotides providedunexpectedly increased apoptosis of cancer cells.

The protocol for the PUMA biomarker was as follows. One day beforetransfection, cells were plated in a 96-well plate at 2×10³ cells perwell with 100 μl of DMEM (HyClone Cat. # SH30243.01) containing 10% FBSand cultured in a 37° C. incubator containing a humidified atmosphere of5% CO2 in air. Next day, before transfection the medium was replacedwith 90 μl of Opti-MEM I Reduced Serum Medium (Life Technologies Cat.#31985-070) containing 2% FBS. Then, 0.2 μl of Lipofectamine RNAiMAX(Life Technologies Cat. #13778-100) were mixed with 4.8 μl of Opti-MEM Ifor 5 minutes at room temperature. 1 μl of the GST-π siRNA (stock conc.1 μM) was mixed with 4 μl of Opti-MEM I and combined with the RNAiMAXsolution and then mixed gently. The mixture was incubated for 10 minutesat room temperature to allow the RNA-RNAiMAX complexes to form. 10 μl ofRNA-RNAiMAX complexes were added per well, to final concentration of thesiRNA 10 nM. The cells were incubated for 2 hours and medium changed tofresh Opti-MEM I Reduced Serum Medium containing 2% FBS. For 1, 2, 3, 4,and 6 days post transfection, the cells were washed with ice-cold PBSonce and then lysed with 50 μl of Cell-to-Ct Lysis Buffer (LifeTechnologies Cat. #4391851 C) for 5-30 minutes at room temperature. 5 μlof Stop Solution was added and incubated for 2 minutes at roomtemperature. PUMA (BBC3, Cat# Hs00248075, Life Technologies) mRNA levelswere measured by qPCR with TAQMAN.

Example 12

The GST-π siRNAs of this invention can exhibit profound reduction ofcancer xenograft tumors in vivo. The GST-π siRNAs can provide geneknockdown potency in vivo when administered in a liposomal formulationto the cancer xenograft tumors.

FIG. 5 shows tumor inhibition efficacy for a GST-π siRNA (SEQ ID NOs:63and 128). Dose dependent knockdown of GST-π mRNA was observed in vivowith the siRNA targeted to GST-π. A cancer xenograft model was utilizedwith a relatively low dose at 0.75 mg/kg of siRNA targeted to GST-π.

The GST-π siRNA showed significant and unexpectedly advantageous tumorinhibition efficacy within a few days after administration. As shown inFIG. 5, treatment with a GST-π siRNA resulted in significant reductionof GST-π mRNA expression 4 days after injection in a lipid formulation.At the higher dose of 4 mg/kg, significant reduction of about 40% wasdetected 24 hours after injection.

The GST-π siRNA was administered in a single injection of 10 mL/kg of aliposomal formulation having the composition (Ionizablelipid:Cholesterol:DOPE:DOPC:DPPE-PEG-2K) (25:30:20:20:5).

For the cancer xenograft model, an A549 cell line was obtained fromATCC. The cells were maintained in RPMI-1640 supplemented with 10% FetalBovine Serum and 100 U/ml penicillin and 100 μg/ml streptomycin. Cellswere split 48 hrs before inoculation so that cells were in log phasegrowth when harvested. Cells were lightly trypsinized with trypsin-EDTAand harvested from tissue culture. The number of viable cells wascounted and determined in a hemocytometer in the presence of trypan blue(only viable cells are counted). The cells were resuspended to aconcentration of 4×10⁷/ml in RPMI media without serum. Then the cellsuspension was mixed well with ice thawed BD matrigel at 1:1 ratio forinjection.

Mice were Charles River Laboratory Athymic Nude (nu/nu) Female Mice,immuno-compromised, 6-8 weeks old, 3 mice per group.

For tumor model preparation, each mouse was inoculated subcutaneously inthe right flank with 0.1 ml an inoculum of 2×10⁶ of A549 cells using a25 G needle and syringe, one inoculum per mouse. Mice were notanesthetized for inoculation.

For tumor volume measurements and randomization, tumor size was measuredto the nearest 0.1 mm. Tumor volumes were calculated using the formula:Tumor volume=length×width/2. Tumor volumes were monitored twice a week.Once the established tumors reached approximately 350-600 mm³, the micewere assigned into groups with varied time points. On the same day, testarticles were administered according to the dosing regimen.

For dosage administration, on the day when the established tumorsreached approximately 350-600 mm³, the test articles were taken out from4° C. fridge. Before being applied to syringes, the bottle containingformulation was reverted by hand for a few times to make a homogeneoussolution.

For body weight, mice were weighed to the nearest 0.1 g. Body weightswere monitored and recorded twice for weeks, for the rest of weeks,including the day of study termination.

For tumors collection, animals were sacrificed by overdosed CO₂ andtumors were dissected at 0, 24, 48, 72, 96 (optional), and 168 hoursfollowing the dosing.

Tumors were first wet weighted, and then separated into three parts forKD, distribution and biomarker analysis. The samples were snap frozen inliquid nitrogen and stored at −80° C. until ready to be processed.

Example 13

The GST-π siRNAs of this invention inhibited pancreatic cancer xenografttumors in vivo. The GST-π siRNAs provided gene knockdown potency in vivowhen administered in a liposomal formulation to the pancreatic cancerxenograft tumors.

In this xenograft model, each mouse was inoculated subcutaneously in theright flank with 0.1 ml an inoculum of 2.5×10⁶ of PANC-1 cells. Athymicnude female mice, 6 to 8 weeks, Charles River, were used. Tumor size wasmeasured to the nearest 0.1 mm. Once the established tumors reachedapproximately 150-250 mm³ (average tumor volume at about 200 mm³), themice were assigned into the various vehicle control and treatment groupssuch that the mean tumor volumes in the treated groups were within 10%of the mean tumor volume in the vehicle control group. On the same day,test articles and control vehicle were administered according to thedosing regimen. Tumor volumes were monitored three times for week 1,twice for the rest of weeks, including the day of study termination.

FIG. 6 shows tumor inhibition efficacy for a GST-π siRNA (SEQ ID Nos:63and 128). As shown in FIG. 6, a dose response was obtained with dosesranging from 0.375 mg/kg to 3 mg/kg of siRNA targeted to GST-π. TheGST-π siRNA showed significant and unexpectedly advantageous tumorinhibition efficacy within a few days after administration. Thus, theGST-π siRNA demonstrated significant and unexpectedly advantageous tumorinhibition efficacy at the endpoint.

The GST-π siRNAs were administered in a liposomal formulation having thecomposition (Ionizable lipid:cholesterol:DOPE:DOPC:DPPE-PEG-2K)(25:30:20:20:5).

Example 14

The GST-π siRNAs of this invention exhibited increased serum stability.

FIG. 7 shows incubation in human serum and detection of remaining siRNAat various time points by HPLS/LCMS. As shown in FIG. 7, the half-life(t_(1/2)) in serum for both the sense strand (FIG. 7, top) and antisensestrand (FIG. 7, bottom) of a GST-π siRNA (SEQ ID Nos:63 and 128) wasabout 100 minutes.

Example 15

The GST-π siRNAs of this invention exhibited enhanced stability informulation in plasma.

FIG. 8 shows incubation of formulation in plasma and detection ofremaining siRNA at various time points. As shown in FIG. 8, thehalf-life (t_(1/2)) in plasma of a formulation of GST-π siRNA (SEQ IDNos:63 and 128) was significantly longer than 100 hours.

The GST-π siRNA was prepared in a liposomal formulation having thecomposition (Ionizing lipid:cholesterol:DOPE:DOPC:DPPE-PEG-2K)(25:30:20:20:5). The z-average size for the liposomal nanoparticles was40.0 nm, and the siRNA was 91% encapsulated.

The formulation was incubated in 50% human serum in PBS for 40 min, 1.5h, 3 h, 24 h, and 96 h. The amount of the GST-π siRNA was determined byan ELISA-based assay.

Example 16

The GST-π siRNAs of this invention exhibited reduced off target effectsby the passenger strand.

For the GST-π siRNA (SEQ ID Nos:158 and 184), FIG. 9 shows that in vitroknockdown for the guide strand was approximately exponential, ascompared to a control with scrambled sequence that exhibited no effect.The IC50 of this siRNA was measured at 5 pM. FIG. 10 shows in vitroknockdown for the passenger strand of the same GST-π siRNA. As shown inFIG. 10, the passenger strand off target knockdown for the GST-π siRNAwas greatly reduced, by more than 100-fold.

For the GST-π siRNAs (SEQ ID Nos:189 and 201), (SEQ ID Nos:191 and 203),and (SEQ ID Nos:192 and 204), FIG. 11 shows that the in vitro knockdownsfor the guide strands were approximately exponential. The IC50s of thesesiRNAs were measured at 6, 7, and 5 pM, respectively. As shown in FIG.12, the in vitro knockdowns for the passenger strands of these GST-πsiRNAs were significantly reduced by at least 10-fold. All of theseGST-π siRNAs had deoxynucleotides in the seed region of the duplexregion, with no other modifications in the duplex region.

For the GST-π siRNAs (SEQ ID Nos:219 and 234), FIG. 13 shows that the invitro knockdown for the guide strand of this highly active GST-π siRNAwas approximately exponential. The IC50 of this siRNA was measured at 11pM. As shown in FIG. 14, the in vitro knockdown for the passenger strandof this GST-π siRNA was significantly reduced by more than 100-fold.This GST-π siRNA had deoxynucleotides in the seed region of the duplexregion, with no other modifications in the duplex region.

Off-target effects were determined using the expression reporter plasmidpsiCHECK-2, which encodes the Renilla luciferase gene. (Dual-LuciferaseReporter Assay System, Promega, Cat#:E1960). The siRNA concentration wastypically 50 pM. Protocol: Day 1, HeLa cell seeded at 5 to 7.5×103/100ul/well. Day 2, co-transfection with cell confluence about 80%. Day 3,cells harvested for luciferase activity measurement. Luciferase activitywas measured using Promega's Luciferase Assay System (E4550), accordingto manufacturer's protocol.

The psiCHECK-2 vector enabled monitoring of changes in expression of atarget gene fused to the reporter gene of Renilla luciferase. The siRNAconstructs were cloned into the multiple cloning region, and the vectorwas cotransfected with the siRNA into HeLa cells. If a specific siRNAbinds to the target mRNA and initiates the RNAi process, the fusedRenilla luciferase: construct mRNA will be cleaved and subsequentlydegraded, decreasing the Renilla luciferase signal.

For example, the plasmid inserts for siRNAs with the BU2′ structure wereas follows:

PsiCHECK-2 (F) plasmid insert:

SEQ ID NO.: 288 ctcgag gggcaacTGAAGCCTTTTGAGACCCTGcTgTcccag gcggccgc

PsiCHECK-2 (R) plasmid insert:

SEQ ID NO.: 289 ctcgag cTgggacagCAGGGTCTCAAAAGGCTTCagTTgccc gcggccgc

Example 17

The GST-π siRNAs of this invention exhibited advantageously reducedmiRNA-like off target effects, which are seed-dependent unintendedoff-target gene silencing.

For the GST-π siRNAs (SEQ ID Nos:158 and 184), (SEQ ID Nos:189 and 201),(SEQ ID Nos:191 and 203), (SEQ ID Nos:192 and 204), and (SEQ ID Nos:219and 234), off target activity mimicking miRNA was found to beessentially negligible. The seed-dependent unintended off-target genesilencing for these GST-π siRNAs was at least 10-fold to 100-fold lessthan the on-target activity of the guide strand.

For testing miRNA-related off target effects, one to four repeats ofseed-matched target sequences complementary to the entireseed-containing region, positions 1-8 of the 5′ end of the antisensestrand, but not to the remaining non-seed region, positions 9-21, wereintroduced into the region corresponding to the 3′UTR of the luciferasemRNA, to determine the efficiency of the seed-dependent unintendedoff-target effects. Plasmid inserts were used to mimic a miRNA withcomplete matching in the seed region and mismatches (bulges) in thenon-seed region.

For example, the plasmid inserts for siRNAs with the BU2′ structure wereas follows:

PsiCHECK-2 (Fmi1) plasmid insert:

SEQ ID NO.: 290 ctcgag gggcaacTCTACGCAAAACAGACCCTGcTgTcccag gcggccgc

PsiCHECK-2 (Fmi2) plasmid insert:

SEQ ID NO.: 291 ctcgag gggcaacTCTACGCAAAACAGACCCTGcTCTACGCAAAACAGACCCTGcT gTcccag gcggccgc

PsiCHECK-2 (Fmi3) plasmid insert:

SEQ ID NO.: 292 ctcgag gggcaacTCTACGCAAAACAGACCCTGcTCTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT gTcccag gcggccgc

PsiCHECK-2 (Fmi4) plasmid insert:

SEQ ID NO.: 293 ctcgag gggcaacTCTACGCAAAACAGACCCTGcTCTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcTgTcccag gcggccgc

Example 18

Examples of RNAi molecules of this invention targeted to GST-π mRNA areshown in Table 18.

TABLE 18 RNAi molecule sequences for GST-π SENSE STRAND ANTISENSE STRANDID SEQ ID (5′-->3′) SEQ ID (5′-->3′) (A9) NO SEQ ID NOS: 294 to 297 NOSEQ ID NOS: 298 to 301 21 294 CCUUUUGAGACCCUGCUGUUU 298ACAgCaGgGUCUCAAAAGGUU 22 295 CCUUUUGAGACCCUGCUGUUU 299ACAgCaGgGUCUCAAAAGGUU 23 296 CCUUUUGAGACCCUGCUGUUU 300ACAgCaGgGUCUCAAAAGGUU 24 297 CCUUUUGAGACCCUGCUGUUU 301ACAgCaGgGUCUCAAAAGGUU Key for Table 18: Upper case A, G, C and U referto ribo-A, ribo-G, ribo-C and ribo-U, respectively. The lower caseletters a, u, g, c, t refer to 2′-deoxy-A, 2′-deoxy-U, 2′-deoxy-G,2′-deoxy-C, and deoxythymidine (dT = T = t) respectively. Underliningrefers to 2′-OMe-substituted, e.g., U.

The structure of GST-π siRNAs of Table 18 provided unexpectedlyincreased gene knockdown activity in vitro. In vitro transfection wasperformed in an A549 cell line to determine knockdown efficacy for GST-πsiRNAs of Table 18, which are based on structure A9. Dose dependentknockdown of GST-π mRNA was observed with the GST-π siRNAs of Table 18,as shown in Table 19.

TABLE 19 Dose dependent knockdown of GST-π mRNA in an A549 cell line forGST-π siRNAs based on structure B13′ GST-π siRNA structure IC50 (pM) A9(SEQ ID NOs: 294 and 298) 4 A9 (SEQ ID NOs: 295 and 299) 28 A9 (SEQ IDNOs: 296 and 300) 12 A9 (SEQ ID NOs: 297 and 301) 7

The structure of GST-π siRNAs of Table 18 provided unexpectedlyincreased tumor inhibition efficacy in vivo. FIG. 15 shows tumorinhibition efficacy in vivo for GST-π siRNAs having structure based onsiRNA A9: (SEQ ID NOs:294 and 298) and (SEQ ID NOs:297 and 301). Acancer xenograft model using A549 cells was utilized with a relativelylow dose of siRNA at 0.5 mg/kg. The GST-π siRNAs showed advantageoustumor inhibition within a few days. After 36 days, the GST-π siRNAsshowed markedly advantageous tumor inhibition, with final tumor averagevolumes significantly reduced by about 2-fold, as compared to control.

Example 19

Examples of RNAi molecules of this invention targeted to GST-π mRNA areshown in Table 20.

TABLE 20 RNAi molecule sequences for GST-π SEQ SENSE STRAND SEQANTISENSE STRAND ID ID (5′-->3′) ID (5′-->3′) (B13) NO SEQ ID NOS: 302to 303 NO SEQ ID NOS: 304 to 305 21 302 GGAUGACUAUGUGAAGGCAUU 304UGCcUuCaCAUAGUCAUCCUU 22 303 GGAUGACUAUGUGAAGGCAUU 305UGCcUuCaCAUAGUCAUCCUU Key for Table 20: Upper case A, G, C and U referto ribo-A, ribo-G, ribo-C and ribo-U, respectively. The lower caseletters a, u, g, c, t refer to 2′-deoxy-A, 2′-deoxy-U, 2′-deoxy-G,2′-deoxy-C, and deoxythymidine (dT = T = t) respectively. Underliningrefers to 2′-OMe-substituted, e.g., U.

The structure of GST-π siRNAs of Table 18 provided unexpectedlyincreased gene knockdown activity in vitro. In vitro transfection wasperformed in an A549 cell line to determine knockdown efficacy for GST-πsiRNAs of Table 20, which are based on structure B13. Dose dependentknockdown of GST-π mRNA was observed with the GST-π siRNAs of Table 20,as shown in Table 21.

TABLE 21 Dose dependent knockdown of GST-π mRNA in an A549 cell line forGST-π siRNAs based on structure B13′ GST-π siRNA structure IC50 (pM) B13(SEQ ID NOs: 302 and 304) 6 B13 (SEQ ID NOs: 303 and 305) 5

The structure of GST-π siRNAs of Table 20 provided unexpectedlyincreased tumor inhibition efficacy in vivo. FIG. 16 shows tumorinhibition efficacy in vivo for a GST-π siRNA having structure based onsiRNA B13: (SEQ ID NOs:303 and 305). A cancer xenograft model using A549cells was utilized with a relatively low dose of siRNA at 0.75 mg/kg ina formulation with HEPES buffer. The GST-π siRNA showed advantageoustumor inhibition within a few days. After 36 days, the GST-π siRNAshowed markedly advantageous tumor inhibition, with final tumor averagevolumes significantly reduced by about 2-fold, as compared to control.

Additional Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used, and no special significance isto be placed upon whether or not a term is elaborated upon, or discussedherein. The descriptions of examples in this disclosure are illustrativeonly, and in no way limit the scope and meaning of the invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. The following references canprovide a general definition of certain terms used in this invention:Singleton et al., Dictionary of Microbiology and Molecular Biology (2nded. 1994); The Cambridge Dictionary of Science and Technology (Walkered., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.),Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionaryof Biology (1991).

A “neoplasia” can refer to any disease that is caused by, or results ininappropriately high levels of cell division, inappropriately low levelsof apoptosis, or both. For example, cancer is an example of a neoplasia.Examples of cancers include leukemias, e.g., acute leukemia, acutelymphocytic leukemia, acute myelocytic leukemia, acute myeloblasticleukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia,acute monocytic leukemia, acute erythroleukemia, chronic leukemia,chronic myelocytic leukemia, chronic lymphocytic leukemia, polycythemiavera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors such assarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioepdotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma).Lymphoproliferative disorders are also considered to be proliferativediseases.

By “nucleic acid” is meant an oligomer or polymer of ribonucleic acid ordeoxyribonucleic acid, or analog thereof. This term includes oligomersconsisting of naturally occurring bases, sugars, and intersugar(backbone) linkages as well as oligomers having non-naturally occurringportions which function similarly. Such modified or substitutedoligonucleotides are often preferred over native forms because ofproperties such as, for example, enhanced stability in the presence ofnucleases.

By “substantially identical” is meant a protein or nucleic acid moleculeexhibiting at least 50% identity to a reference amino acid sequence (forexample, any one of the amino acid sequences described herein) ornucleic acid sequence (for example, any one of the nucleic acidsequences described herein). Preferably, such a sequence is at least60%, more preferably 80% or 85%, and still more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

By “inhibitory nucleic acid” is meant a single or double-stranded RNA,siRNA (short interfering RNA), shRNA (short hairpin RNA), or antisenseRNA, or a portion thereof, or a mimetic thereof, that when administeredto a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%,or even 90-100%) in the expression of a target gene. Typically, anucleic acid inhibitor comprises or corresponds to at least a portion ofa target nucleic acid molecule, or an ortholog thereof, or comprises atleast a portion of the complementary strand of a target nucleic acidmolecule.

By “antisense nucleic acid”, it is meant a non-enzymatic nucleic acidmolecule that binds to target RNA by means of RNA-RNA or RNA-DNAinteractions and alters the activity of the target RNA (for a review,see Stein et al. 1993; Woolf et al., U.S. Pat. No. 5,849,902).Typically, antisense molecules are complementary to a target sequencealong a single contiguous sequence of the antisense molecule. However,in certain embodiments, an antisense molecule can bind to substrate suchthat the substrate molecule forms a loop, and/or an antisense moleculecan bind such that the antisense molecule forms a loop. Thus, theantisense molecule can be complementary to two (or even more)non-contiguous substrate sequences or two (or even more) non-contiguoussequence portions of an antisense molecule can be complementary to atarget sequence or both. For a review of current antisense strategies,see Schmajuk N A et al., 1999; Delihas N et al., 1997; Aboul-Fadl T,2005.)

The term “siRNA” refers to small interfering RNA; a siRNA is a doublestranded RNA that “corresponds” to or matches a reference or target genesequence. This matching need not be perfect so long as each strand ofthe siRNA is capable of binding to at least a portion of the targetsequence. siRNAs can be used to inhibit gene expression, see for exampleBass, 2001, Nature, 411, 428 429; Elbashir et al., 2001, Nature, 411,494 498; and Zamore et al., Cell 101:25-33 (2000).

The embodiments described herein are not limiting and one skilled in theart can readily appreciate that specific combinations of themodifications described herein can be tested without undueexperimentation toward identifying nucleic acid molecules with improvedRNAi activity.

All publications, patents and literature specifically mentioned hereinare incorporated by reference in their entirety for all purposes.

It is understood that this invention is not limited to the particularmethodology, protocols, materials, and reagents described, as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention. It will be readilyapparent to one skilled in the art that varying substitutions andmodifications can be made to the description disclosed herein withoutdeparting from the scope and spirit of the description, and that thoseembodiments are within the scope of this description and the appendedclaims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprises,” “comprising”,“containing,” “including”, and “having” can be used interchangeably, andshall be read expansively and without limitation.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. For Markush groups, those skilled in theart will recognize that this description includes the individualmembers, as well as subgroups of the members of the Markush group.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever.

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose.

What is claimed is:
 1. A pharmaceutical composition for the treatment ortherapy of a malignant tumor, the composition comprising RNAi moleculesand pharmaceutically acceptable excipients, wherein the RNAi moleculescomprise a sense strand which comprises CCUUUUGAGACCCUGCUGUUU (SEQ IDNO:294) and an antisense strand which comprises ACAgCaGgGUCUCAAAAGGUU(SEQ ID NO:298), or wherein the RNAi molecules comprise a sense strandwhich comprises GGAUGACUAUGUGAAGGCAUU (SEQ ID NO:303) and an antisensestrand which comprises UGCcUuCaCAUAGUCAUCCUU (SEQ ID NO:305), wherein anuppercase letter refers to an unmodified ribonucleotide, wherein a lowercase letter refers to 2′-deoxy, and wherein the underlined nucleotidesare 2′-OMe-substituted.
 2. The pharmaceutical composition of claim 1,wherein the RNAi molecules are siRNAs or shRNAs.
 3. The pharmaceuticalcomposition of claim 1, wherein the pharmaceutically acceptableexcipients include one or more lipid compounds.
 4. The pharmaceuticalcomposition of claim 1, wherein the pharmaceutically acceptableexcipients include lipid nanoparticles.
 5. The pharmaceuticalcomposition of claim 1, wherein the pharmaceutically acceptableexcipients include lipid nanoparticles that encapsulate the RNAimolecules.
 6. A method for preventing, treating or ameliorating one ormore symptoms of a malignant tumor associated with KRAS mutation in amammal in need thereof, the method comprising: identifying a tumor cellin the mammal, the tumor cell comprising at least one of: (i) a mutationof the KRAS gene, and (ii) an aberrant expression level of KRAS protein;and administering to the mammal a therapeutically effective amount of acomposition comprising one or more RNAi molecules that are active inreducing expression of GST-π; wherein the RNAi molecules comprise asense strand which comprises CCUUUUGAGACCCUGCUGUUU (SEQ ID NO:294) andan antisense strand which comprises ACAgCaGgGUCUCAAAAGGUU (SEQ IDNO:298), or wherein the RNAi molecules comprise a sense strand whichcomprises GGAUGACUAUGUGAAGGCAUU (SEQ ID NO:303) and an antisense strandwhich comprises UGCcUuCaCAUAGUCAUCCUU (SEQ ID NO:305), wherein anuppercase letter refers to an unmodified ribonucleotide, wherein a lowercase letter refers to 2′-deoxy, and wherein the underlined nucleotidesare 2′-OMe-substituted.
 7. The method of claim 6, wherein the mammal isa human and the GST-π is a human GST-π.
 8. The method of claim 6,wherein the RNAi molecule is a siRNA or shRNA.
 9. The method of claim 6,wherein the administration decreases expression of GST-π in the mammal.10. The method of claim 6, wherein the administration decreasesexpression of GST-π in the mammal by at least 5% for at least 5 days.11. The method of claim 6, wherein the method reduces one or moresymptoms of the malignant tumor, or delays or terminates the progressionof the malignant tumor.
 12. The method of claim 6, wherein theadministration reduces growth of malignant tumor cells in the subject.13. The method of claim 6, wherein the administration reduces growth forat least 10% of the malignant tumor cells in the subject.
 14. The methodof claim 6, wherein the tumor cells comprise increased levels ofexpression of wild type KRAS protein compared to that in a normal cell.15. The method of claim 6, wherein the tumor cell over-expresseswild-type GST-π RNA or protein.
 16. The method of claim 6, wherein thetumor cell comprises mutations in the KRAS protein at one or more ofresidues 12, 13 and
 61. 17. The method of claim 6, wherein the tumorcell comprises mutations in the KRAS protein and the tumor is a cancerselected from lung cancer, colon cancer, and pancreatic cancer.
 18. Themethod of claim 6, wherein the tumor cell comprises mutations in theKRAS protein and the tumor is a sarcoma or carcinoma selected from thegroup consisting of lung adenocarcinoma, mucinous adenoma, ductalcarcinoma of the pancreas, and colorectal carcinoma.
 19. The method ofclaim 6, wherein the malignant tumor is a sarcoma or carcinoma selectedfrom the group of lung adenocarcinoma, mucinous adenoma, ductalcarcinoma of the pancreas, colorectal carcinoma, breast cancer, andfibrosarcoma.
 20. The method of claim 6, wherein the malignant tumor islocated in an anatomical region selected from the group of lung, colon,pancreas, gallbladder, liver, breast, and any combination thereof. 21.An RNAi molecule comprising a sense strand which comprisesCCUUUUGAGACCCUGCUGUUU (SEQ ID NO:294) and an antisense strand whichcomprises ACAgCaGgGUCUCAAAAGGUU (SEQ ID NO:298), wherein an uppercaseletter refers to an unmodified ribonucleotide, wherein a lower caseletter refers to 2′-deoxy, and wherein the underlined nucleotides are2′-OMe-substituted.
 22. An RNAi molecule comprising a sense strand whichcomprises GGAUGACUAUGUGAAGGCAUU (SEQ ID NO:303) and an antisense strandwhich comprises UGCcUuCaCAUAGUCAUCCUU (SEQ ID NO:305), wherein anuppercase letter refers to an unmodified ribonucleotide, wherein a lowercase letter refers to 2′-deoxy, and wherein the underlined nucleotidesare 2′-OMe-substituted.